U.S. patent number 11,168,084 [Application Number 16/760,727] was granted by the patent office on 2021-11-09 for purine based compounds as toll-like receptor 9 antagonist.
This patent grant is currently assigned to Council of Scientific & Industrial Research. The grantee listed for this patent is COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH. Invention is credited to Raychaudhuri Deblina, Dipyaman Ganguly, Biswajit Kundu, Ayan Mukherjee, Barnali Paul, Oindrila Rahaman, Swarnali Roy, Arindam Talukdar.
United States Patent |
11,168,084 |
Talukdar , et al. |
November 9, 2021 |
Purine based compounds as toll-like receptor 9 antagonist
Abstract
The present invention provides novel purine based compounds of
formula 1, method of preparation of purine based compounds and its
composition useful for inhibiting signalling through Toll-like
receptors. These compounds are useful in inhibiting immune
stimulation involving toll-like receptor 9 (TLR9). These can be
used in treatment of autoimmune disease and inflammation where
aberrant activation of TLR9 plays role. ##STR00001##
Inventors: |
Talukdar; Arindam (Kolkata,
IN), Ganguly; Dipyaman (Kolkata, IN),
Mukherjee; Ayan (Kolkata, IN), Paul; Barnali
(Kolkata, IN), Rahaman; Oindrila (Kolkata,
IN), Kundu; Biswajit (Kolkata, IN), Roy;
Swarnali (Kolkata, IN), Deblina; Raychaudhuri
(Kolkata, IN) |
Applicant: |
Name |
City |
State |
Country |
Type |
COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH |
New Delhi |
N/A |
IN |
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Assignee: |
Council of Scientific &
Industrial Research (New Delhi, IN)
|
Family
ID: |
1000005923935 |
Appl.
No.: |
16/760,727 |
Filed: |
November 5, 2018 |
PCT
Filed: |
November 05, 2018 |
PCT No.: |
PCT/IN2018/050714 |
371(c)(1),(2),(4) Date: |
April 30, 2020 |
PCT
Pub. No.: |
WO2019/092739 |
PCT
Pub. Date: |
May 16, 2019 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200347062 A1 |
Nov 5, 2020 |
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Foreign Application Priority Data
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Nov 8, 2017 [IN] |
|
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201711039774 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D
473/16 (20130101) |
Current International
Class: |
C07D
473/16 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2012167053 |
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Dec 2012 |
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WO |
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2017163264 |
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Sep 2017 |
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WO |
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Other References
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of TLR7 and TLR9 leads to reduction of autoantibody production and
melioration of disease symptoms", Eur. J. Immunol. vol. 37, pp.
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Graft-versus-Host Disease", The Journal of Immunology, vol. 1818,
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via Toll-like Receptor 7 and 8", Science, vol. 303, pp. 1526-1529,
Mar. 5, 2004. cited by applicant .
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.
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Death With Inflammation in Acute Pancreatitis", Gastroenterology,
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Through Cross-Talk with Neutrophils", Shock, vol. 36, No. 6, pp.
548-552, 2011. cited by applicant .
Lande et al., "Plasmacytoid dendritic cells sense self-DNA coupled
with anticicrobial peptide", Nature, vol. 449, pp. 564-571, Oct. 4,
2007. cited by applicant .
Lande et al., "Neutrophils Activate Plasmacytoid Dendritic Cells by
Releasing Self-DNA-Peptide Complexes in Systemic Lupus
Erythematosus", Science Translational Medicine, vol. 3, Issue 73,
pp. 1-11, Mar. 9, 2011. cited by applicant .
Leadbetter et al., "Chromatin-IgG complexes activate B cells by
dual engagement of IgM and Toll-like receptors", Nature, vol. 416,
pp. 603-607, Apr. 11, 2002. cited by applicant .
Lund et al., "Recognition of single-stranced RNA viruses by
Toll-like receptor 7", PNAS, vol. 101, No. 15, pp. 5598-5603, Apr.
13, 2004. cited by applicant .
Marshak-Rothstein, "Toll-like receptors in systemic autoimmune
disease", Naqture Reviews/Immunology, vol. 6, pp. 823-835, Nov.
2006. cited by applicant .
Medzhitov et al., "A human homologue of the Drosophila Toll protein
signals activation of adaptive immunity", Nature, vol. 388, pp.
394-397, Jul. 24, 1997. cited by applicant .
Medzhitov, "Toll-like Receptors and Innate Immunity", Nature, vol.
1, pp. 135-145, Nov. 2001. cited by applicant .
Takeda et al., "Toll-Like Receptors", Annu. Rev. Immunol, vol. 21,
pp. 335-376, 2003. cited by applicant .
Search Report and Written Opinion pertaining to PCT/IN2018/050714
dated Jan. 30, 2019. cited by applicant.
|
Primary Examiner: Anderson; Rebecca L
Attorney, Agent or Firm: Dinsmore & Shohl LLP
Claims
We claim:
1. A compound of Formula 1 or pharmaceutically acceptable salts
thereof, ##STR00114## wherein R.sub.1 is independently chosen from:
##STR00115## wherein R.sub.2 is independently chosen from:
##STR00116## wherein R.sub.3 is independently chosen from:
##STR00117##
2. The compound of Formula 1 as claimed in claim 1, wherein the
compound is selected from the group consisting of:
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-2--
amine (compound 4);
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4-methox-
ybenzyl)-9H-purin-2-amine (compound 5);
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4-fluoro-
benzyl)-9H-purin-2-amine (compound 6);
N-(4-(diethylamino)benzyl)-6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperaz-
in-1-yl)propyl)-9H-purin-2-amine (compound 7);
N-benzyl-6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purin-2-amine (compound 8);
6-(4-ethylpiperazin-1-yl)-9(3-(4-ethylpiperazin-1-yl)propyl-2-(pyrrolidin-
-1-yl)-9H-purine (compound 9);
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)benzamide (compound 10);
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl) pyrrolidine-2-carboxamide (compound 12);
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl) benzenesulfonamide (compound 13);
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)acetamide (compound 14); Iso-butyl
(1-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-puri-
n-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (compound 15);
4-((dimethylamino)methyl)-N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiper-
azin-1-yl)propyl)-9H-purin-2-yl)benzamide (compound 16);
(4-(2-amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-6-yl)piperazin-1-
-yl)(phenyl)methanone (compound 18);
4-(2-((4-(diethylamino)benzyl)amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9-
H-purine-6-yl)piperazin-1-yl)(phenyl)methanone (compound 19);
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-(pyrrolidin-1-yl)-9H-purin-6-yl-
)piperazin-1-yl)(phenyl)methanone (compound 20);
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-methoxybenzyl)amino)-9H-pur-
in-6-yl)piperazin-1-yl)(phenyl)methanone (compound 21);
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-fluorobenzyl)amino)-9H-puri-
n-6-yl)piperazin-1-yl)(phenyl)methanone (compound 22);
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-pu-
rin-2-amine (compound 23);
6-(4-cyclopentylpiperazin-1-yl)-N-(4-diethylamino)benzyl)-9-(3-(4-ethylpi-
perazine-1-yl)propyl)-9H-purine-2-amine (compound 24);
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4--
fluorobenzyl)-9H-purin-2-amine (compound 25);
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4--
methoxybenzyl)-9H-purin-2-amine (compound 26);
N-(6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purin-2-yl)-4-((dimethylamino)methyl)benzamide (compound 27);
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-2--
amine (compound 29);
6-(4-cyclopentylpiperazin-1-yl)-N-(4-methoxybenzyl)-9-(3-(pyrrolidin-1-yl-
)propyl)-9H-purin-2-amine (compound 30);
N-(6-(4-cyclopentylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-
-2-yl)-4-((dimethylamino)methyl)benzamide (compound 31);
6-(4-cyclopentylpiperazin-1-yl)-N-(4-fluorobenzyl)-9-(3-(pyrrolidin-1-yl)-
propyl)-9H-purin-2-amine (compound 32);
6-(4-ethylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-2-amine
(compound 33);
6-(4-ethylpiperazin-1-yl)-N-(4-methoxybenzyl)-9-(3-(pyrrolidin-1-yl)propy-
l)-9H-purin-2-amine (compound 34);
4-((dimethylamino)methyl)-N-(6-(4-ethylpiperazin-1-yl)-9-(3-(pyrrolidin-1-
-yl)propyl)-9H-purin-2-yl)benzamide (compound 35);
6-(4-ethylpiperazin-1-yl)-N-(4-fluorobenzyl)-9-(3-(pyrrolidin-1-yl)propyl-
)-9H-purin-2-amine (compound 36);
(4-(2-Amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-6-yl)piperazin-1-
-yl)(cyclopentyl)methanone (compound 37);
Cyclopentyl(4-(2-(4-(diethylamino)benzylamino)-9-(3-(4-ethylpiperazin-1-y-
l)propyl)-9H-purin-6-yl)piperazin-1-yl)methanone (compound 38);
Cyclopentyl(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-(4-methoxybenzylamin-
o)-9H-purin-6-yl)piperazin-1-yl)methanone (compound 39); and
Cyclopentyl(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-fluorobenzyl)ami-
no)-9H-purin-6-yl)piperazin-1-yl)methanone (compound 40).
3. A method of treating an auto-immune disease associated with TLR9
activation in a subject in need thereof, the method comprising
providing an effective amount of the compound of claim 1 to the
subject.
4. A method of inhibiting TLR9-mediated immune-stimulatory
signaling, the method comprising contacting a cell expressing TLR9
with an effective amount of the compound of claim 1.
5. A process for preparation of the compound of Formula 1 of claim
1, the method comprising: (i) reacting 6-chloro-9H-purin-2-amine,
(compound 1), with ethyl piperazine or
1-(3-chloropropyl)-4-ethylpiperazine in presence of a base in a
solvent at reflux temperature for a period ranging between 3 to 4
hrs to obtain 6-(4-ethylpiperazine-1-yl)-9H-purin-2-amine (compound
2) or 6-chloro-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-2-amine
(compound 17); (ii) reacting compound 2 with 1-bromo-3-chloro
propane in presence of a base in DMSO, DMF at room temperature for
a period ranging between 10 to 12 hrs to obtain
9-(3-chloropropyl)-6-(4-ethylpiperazine-1-yl)-9H-purin-2-amine
(compound 3); (iii)reacting compound 3 with ethyl piperazine or
pyrrolidine in presence of a base DIPEA or Et.sub.3N in a solvent
to obtain
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-2--
amine (compound 4) or
6-(4-ethylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-2-amine
(compound 33) respectively; (iv)reacting compound 4 as obtained in
(iii) with a compound selected from the group consisting of
aromatic aldehyde, acid, acid chloride, and dibromoalkane in
presence of a base and a solvent at a temperature range between 25
to 110.degree. C., for a period ranging between 3 to 24 hrs to
obtain: a.
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4-methox-
ybenzyl)-9H-purin-2-amine (compound 5); b.
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4-fluoro-
benzyl)-9H-purin-2-amine (compound 6); c.
N-(4-(diethylamino)benzyl)-6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperaz-
in-1-yl)propyl)-9H-purin-2-amine (compound 7); d.
N-benzyl-6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purin-2-amine (compound 8); e.
6-(4-ethylpiperazin-1-yl)-9(3-(4-ethylpiperazin-1-yl)propyl-2-(pyrrolidin-
-1-yl)-9H-purine (compound 9); f.
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)benzamide (compound 10); g. tert-butyl
2-((6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-puri-
n-2-yl)carbamoyl)pyrrolidine-1-carboxylate (compound 11), h.
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)pyrrolidine-2-carboxamide (compound 12); i.
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)benzenesulfonamide (compound 13); j.
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)acetamide (compound 14); k. Iso-butyl
(1-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-puri-
n-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (compound 15); or
l.
4-((dimethylamino)methyl)-N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiper-
azin-1-yl)propyl)-9H-purin-2-yl)benzamide (compound 16); (v)
reacting compound 1 or compound 17 with a compound phenyl
(piperazine-1-yl)methanone, 1-cyclopentylpiperazine, or
cyclopentyl(piperazin-1-yl)methanone in presence of a base selected
from Cs.sub.2CO.sub.3 or K.sub.2CO.sub.3 in a solvent selected from
THF, dioxane, or CH.sub.3CN at a temperature ranging between 80 to
100.degree. C. to obtain: a.
(4-(2-amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-6-yl)piperazin-1-
-yl)(phenyl)methanone (compound 18); b.
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-pu-
rin-2-amine (compound 23); c.
6-(4-cyclopentylpiperazin-1-yl)-9H-purin-2-amine (compound 28); or
d.
(4-(2-Amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-6-yl)piperazin-1-
-yl)(cyclopentyl)methanone (compound 37); (vi)reacting compound 18
or compound 23 with a compound selected from the group consisting
of aldehyde, acid and dibromo alkane in a solvent and in the
presence of a base at a temperature ranging between 80 to
110.degree. C. for a period ranging between 12 to 24 hrs to obtain:
a.
4-(2-((4-(diethylamino)benzyl)amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9-
H-purine-6-yl)piperazin-1-yl)(phenyl)methanone (compound 19); b.
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-(pyrrolidin-1-yl)-9H-purin-6-yl-
)piperazin-1-yl)(phenyl)methanone (compound 20); c.
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-methoxybenzyl)amino)-9H-pur-
in-6-yl)piperazin-1-yl)(phenyl)methanone (compound 21); d.
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-fluorobenzyl)amino)-9H-puri-
n-6-yl)piperazin-1-yl)(phenyl)methanone (compound 22); e.
6-(4-cyclopentylpiperazin-1-yl)-N-(4-diethylamino)benzyl)-9-(3-(4-ethylpi-
perazine-1-yl)propyl)-9H-purine-2-amine (compound 24); f.
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4--
fluorobenzyl)-9H-purin-2-amine (compound 25); g.
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4--
methoxybenzyl)-9H-purin-2-amine (compound 26); or h.
N-(6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purin-2-yl)-4-((dimethylamino)methyl)benzamide (compound 27);
(vii) reacting compound 28 with 1-(3-chloropropyl)pyrrolidine to
give
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-2--
amine (compound 29), and (viii) reacting compound 29, compound 33
or compound 37 with a compound of aldehyde or an acid in presence
of a solvent and a base, at a temperature ranging between 25 to
100.degree. C., for a period ranging between 3 hr to 24 hr to
obtain: a.
6-(4-cyclopentylpiperazin-1-yl)-N-(4-methoxybenzyl)-9-(3-(pyrrolidin-1-yl-
)propyl)-9H-purin-2-amine(compound 30); b.
N-(6-(4-cyclopentylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-
-2-yl)-4-((dimethylamino)methyl)benzamide (compound 31); c.
6-(4-cyclopentylpiperazin-1-yl)-N-(4-fluorobenzyl)-9-(3-(pyrrolidin-1-yl)-
propyl)-9H-purin-2-amine (compound 32); d.
6-(4-ethylpiperazin-1-yl)-N-(4-methoxybenzyl)-9-(3-(pyrrolidin-1-yl)propy-
l)-9H-purin-2-amine (compound 34); e.
4-((dimethylamino)methyl)-N-(6-(4-ethylpiperazin-1-yl)-9-(3-(pyrrolidin-1-
-yl)propyl)-9H-purin-2-yl)benzamide (compound 35); f.
6-(4-ethylpiperazin-1-yl)-N-(4-fluorobenzyl)-9-(3-(pyrrolidin-1-yl)propyl-
)-9H-purin-2-amine (compound 36); g.
Cyclopentyl(4-(2-(4-(diethylamino)benzylamino)-9-(3-(4-ethylpiperazin-1-y-
l)propyl)-9H-purin-6-yl)piperazin-1-yl)methanone (compound 38); h.
Cyclopentyl(4-(2-(4-(diethylamino)benzylamino)-9-(3-(4-ethylpiperazin-1-y-
l)propyl)-9H-purin-6-yl)piperazin-1-yl)methanone (compound 39); or
i.
Cyclopentyl(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-fluorobenzyl)ami-
no)-9H-purin-6-yl)piperazin-1-yl)methanone (compound 40).
6. The process as claimed in claim 5, wherein the base of (i) and
(ii) is selected from the group consisting of Cs.sub.2CO.sub.3 and
K.sub.2CO.sub.3.
7. The process as claimed in claim 5, wherein the solvent of (i) is
selected from the group consisting of acetonitrile, THF, dioxane,
and DMF.
8. The process as claimed in claim 5, wherein the solvent of (iii)
is selected from the group consisting of toluene and DCM.
9. The process as claimed in claim 5, wherein the aromatic
aldehyde, acid, acid chloride, and dibromo alkane of (iv) are
selected from the group consisting of benzaldehyde, p-anisaldehyde,
4-fluorobenzaldehyde, 4-diethylamino benzaldehyde, benzoyl
chloride, 1-(Tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid,
benzenesulphonyl chloride, acetyl chloride,
2-(Isobutoxycarbonyl)amino)-3-phenyl propionic acid dibromopropane,
4-[(Dimethylamino)methyl] benzaldehyde, 1, 4-dibromobutane, and
4-[(Dimethylamino)methyl]benzoic acid.
10. The process as claimed in claim 5, wherein the base of (iv),
(vi) and (viii) are selected from the group consisting of pyridine,
sodium hydride, sodium triacetoxy borohydride, sodium
cyanoborohydride, and sodium borohydride.
11. The process as claimed in claim 5, wherein the solvent of (iv),
(vi) and (viii) is selected from the group consisting of toluene,
dichloromethane, THF, and DMF.
12. A pharmaceutical composition comprising an effective amount of
the compounds of Formula 1 as claimed in claim 1 or
pharmaceutically acceptable salts thereof, individually or in
combination, optionally along with one or more pharmaceutically
acceptable additives, carriers or diluents.
13. The process as claimed in claim 5, wherein the aldehyde of step
(vi) and (viii) is selected from the group consisting of
benzaldehyde, p-anisaldehyde, 4-fluorobenzaldehyde and
4-diethylamino benzaldehyde; and the acid of step (vi) and (viii)
is selected from the group consisting of
1-(Tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid,
2-(Isobutoxycarbonyl)amino)-3-phenyl propionic acid, and
4-[(Dimethylamino) methyl]benzoic acid.
14. The process as claimed in claim 5, wherein the dibromo alkane
of step (vi) is selected from the group consisting of
dibromopropane and 1, 4-dibromobutane.
Description
FIELD OF THE INVENTION
The present invention relates to the preparation of new compounds
with general formula (I) in free form or in pharmaceutically
acceptable salts form for inhibiting signalling by TLR9.
##STR00002##
The invention relates to small molecules where R.sub.1, R.sub.2,
R.sub.3, are as defined in the description, capable of inhibiting
immune response mediated through TLR9.
The present invention further relates to the preparation of new
compounds with general formula (I) without considerable
cytotoxicity in HepG2 (a hepatic epithelial cell line) cells at
concentrations below 100 .mu.M.
BACKGROUND AND PRIOR ART OF THE INVENTION
The innate immunity is comprised of several types of cells
including dendritic cells (DC's), macrophages and monocytes,
polymorph nuclear cells, natural killer (NK) cells, innate lymphoid
cells and natural killer T cells (NKT cells) which detects various
pathogens as well as aberrant host cells with potential for danger
to tissue integrity through specialized receptors like toll-like
receptors. Toll-like receptors (TLRs) are a family of
germline-encoded cell surface pattern recognition molecules
containing an pathogen binding ectodomain (ECD) with 19-25
leucine-rich repeats (LRRs), a transmembrane domain and a
characteristic cytoplasmic domain called the TIR (Toll/IL-1
receptor) domain. TIR domain is responsible for downstream
signalling, whereas LRRs containing 24-29 amino acids are
responsible for ligand recognition and binding. TLRs get triggered
in response to bacterial and fungal infections (Medzhitov, R; Nat.
Rev. Immunol. 1, 135-145, 2001) followed by induction of downstream
signalling, leading to expression of inflammatory genes like those
of the nuclear factor-.kappa.B (NF-.kappa.B) family of
transcription factors and antimicrobial peptides. There are 11
human and 12 miceTLRs have been identified which recognize
different molecular patterns on the pathogens.
Major group of the TLRs are expressed on the cell surface. The
leucine-rich repeats in the ectodomains of these molecules bind to
unique molecular entities on pathogens (PAMPs), which detect and
initiate responses to invading microorganisms (Akira, S; et al.
Annu Rev Immunol. 21, 335-76, 2003). Another group of TLRs
(endosomal TLRs) are located inside the cell within the
endosomal-lysosomal compartments, instead of being expressed on the
cell surface (Akira, S; et al. Annu Rev Immunol. 21, 335-76, 2003).
This group comprises of TLR3, TLR7, TLR8 and TLR9. The endosomal
TLRs are specialized for detecting microbial nucleic acids after
microbes get phagocytosed and reach the endosomal compartments.
The downstream signalling goes through recruitment of intracellular
adaptor molecules such as Myd88 (or the myeloid differentiation
primary-response gene 88), TIRAP (or the TIR-domain containing
adaptor protein), TRIF (or the TIRAP inducing IFN-beta) and TRAM
(or the TRIF-related adaptor molecule). TLR-adaptor molecule
interactions in turn recruit other proteins to the signalling
complex, which initiates multiple downstream signalling pathways,
leading to activation of NFkB or mitogen-activated protein kinases
(MAPKs) or recruitment of the IFN regulatory factors (IRFs). These
different pathways in turn result in the transcription of genes
encoding different cytokines, chemokines, co-stimulatory molecules
or other proteins, thereby sculpting the ensuing immune response
(Akira, S; et al. Annu Rev Immunol. 21, 335-76, 2003).
The intracellular localization of the nucleic acid-recognizing TLRs
(TLR3, 7, 8, 9) is one of the mechanisms that prevent their
spontaneous activation by circulating host-derived nucleic acids
(Barton, G. M; et al. Nat Immunol. 7(1):49-56, 2006), however under
certain pathological conditions the endogenous nucleic acids can
overcome this regulation. It has been previously shown by us and
others that the circulating immune complexes found in sera of
patients suffering from systemic lupus erythematosus (SLE)
typically contain nucleic acids associated with various proteins
such as antibodies, the chromatin-associated protein HMGB1, the
antimicrobial peptide LL37, ribonuclear proteins and others (Lande,
R; et al. Nature, 449(7162), 564-9, 2011; Ganguly, D. et al. Nat
Rev Immunol. 13(8), 566-77, 2013). Our previous studies have also
shown that TLR9, 7 and 8 activation driven by self nucleic acid and
LL37 complexes may also play an important pathogenic role in
Psoriasis (Lande, R; et al. Nature, 449(7162), 564-9, 2007;
Ganguly, D. et al. J Exp Med. 206(9), 1983-94, 2009). These
associated proteins may protect the bound nucleic acid from
degradation and/or facilitate their entry into the cell, as is the
case for Fc receptor-mediated uptake of antibody-nucleic acid
complexes (Ganguly, D. et al. J Exp Med. 206(9), 1983-94, 2009).
Once inside the endolysosomal compartments, the nucleic acid cargo
can then stimulate the intracellular TLRs, priming the immune
system for a cascade of inflammation inciting cytotoxic and/or
humoral response. For example, this cycle of innate immune
recognition, generation of autoreactive antibodies, and consequent
immune complex formation is believed to play critical role in the
pathogenesis of SLE and possibly Sjogren's syndrome (Ganguly, D. et
al. Nat Rev Immunol. 13(8), 566-77, 2013), a finding confirmed in
animal models treated with TLR7 and TLR9-competitive antagonist
oligonucleotides (Barrat, F. J; et al. Eur J Immunol. 37(12),
3582-6, 2007). TLR-mediated pathological responses to nucleic acids
have also been shown to contribute to other pathologies like
psoriasis (Lande R et al, Nature, 2007; Ganguly D et al, J Exp Med,
2009), ischemic liver injury (Bamboat, Z. M; et al. Hepatology,
51(2), 621-32, 2010) lung infection (Itagaki, K; et al. Shock,
36(6), 548-52, 2011), pancreatitis (Hoque, R; et al.
Gastroenterology, 141(1), 358-69, 2011) and graft-versus-host
disease (Calcaterra, C; et al. J Immunol. 181(9), 6132-9,
2008).
In literature there are several reports of small molecule analogues
and derivatives of chloroquine with substituted quinoline and
quinazoline scaffold which can inhibit stimulation of the immune
system. U.S. Pat. Nos. 6,479,504; 7,410,975 B2.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide novel purine
based compounds particularly 6-(piperazin-1-yl)-9H-purin-2-amino
compounds and method for preparation thereof.
Another object of the present invention is to provide a screening
method involving human peripheral blood mononuclear cells to screen
compounds of general formula I against TLR9.
Yet another objective of the present invention is to provide a
method for testing TLR9 antagonism of compounds of general formula
I, in primary human plasmacytoid dendritic cells (pDCs) purified
from human peripheral blood mononuclear cells.
Yet another objective of the present invention is to provide a
method for testing TLR9 antagonism of compounds of general formula
I a reporter assay method involving a cell line expressing TLR9 to
screen compounds of general formula I for TLR9 antagonism.
Yet another objective of the present invention is to correlate the
assays results involving human peripheral blood mononuclear cells,
human primary pDCs and transfected TLR9 cells.
Yet another object of the present invention is to provide
composition and methods of compounds of general formula I with TLR9
antagonistic activity that can modulate immune responses.
Yet another object of the present invention is to provide
composition and methods of compounds of general formula I that can
be used in a number of clinical applications, including as
pharmaceutical agents and methods for treating conditions involving
untoward immune hyperactivity.
Yet another object of the present invention is to provide
composition and methods of compounds of general formula I without
considerable cytotoxicity in HepG2 (a hepatic epithelial cell line)
and SW480 (an intestinal mucosal epithelial cell line) cells at
concentrations below 100 .mu.M.
SUMMARY OF THE INVENTION
Accordingly the present invention provides a compound of formula 1
or salts thereof,
##STR00003## Wherein R.sub.1 is independently selected from groups
referred to as follows:
##STR00004## Wherein R.sub.2 is independently selected from groups
referred to as follows:
##STR00005## Wherein R.sub.3 is independently selected from groups
referred to as follows:
##STR00006##
In an embodiment of the invention wherein represented compounds
comprising.
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-2--
amine (4)
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N--
(4-methoxybenzyl)-9H-purin-2-amine (5)
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4-fluoro-
benzyl)-9H-purin-2-amine (6)
N-(4-(diethylamino)benzyl)-6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperaz-
in-1-yl)propyl)-9H-purin-2-amine (7)
N-benzyl-6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purin-2-amine (8)
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl-2-(pyrrolidi-
n-1-yl)-9H-purine (9)
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)benzamide (10)
Tert-butyl-2-((6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)prop-
yl)-9H-purin-2-yl) carbamoyl)pyrrolidine-1-carboxylate (11)
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl) pyrrolidine-2-carboxamide (12)
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl) benzenesulfonamide (13)
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)acetamide (14)
Iso-butyl(1-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl-
)-9H-purin-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (15)
4-((dimethylamino)methyl)-N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiper-
azin-1-yl)propyl)-9H-purin-2-yl)benzamide (16)
(4-(2-amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-6-yl)piperazin-1-
-yl)(phenyl)methanone (18)
4-(2-((4-(diethylamino)benzyl)amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9-
H-purine-6-yl)piperazin-1-yl)(phenyl)methanone (19)
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-(pyrrolidin-1-yl)-9H-purin-6-yl-
)piperazin-1-yl)(phenyl)methanone (20)
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-methoxybenzyl)amino)-9H-pur-
in-6-yl)piperazin-1-yl)(phenyl)methanone (21)
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-fluorobenzyl)amino)-9H-puri-
n-6-yl)piperazin-1-yl)(phenyl)methanone (22)
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-pu-
rin-2-amine (23)
6-(4-cyclopentylpiperazin-1-yl)-N-(4-diethylamino)benzyl)-9-(3-(4-ethylpi-
perazine-1-yl)propyl)-9H-purine-2-amine (24)
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4--
fluorobenzyl)-9H-purin-2-amine (25)
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4--
methoxybenzyl)-9H-purin-2-amine (26)
N-(6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purin-2-yl)-4-((dimethylamino)methyl)benzamide (27)
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-2--
amine (29)
6-(4-cyclopentylpiperazin-1-yl)-N-(4-methoxybenzyl)-9-(3-(pyrro-
lidin-1-yl)propyl)-9H-purin-2-amine (30)
N-(6-(4-cyclopentylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-
-2-yl)-4-((dimethylamino)methyl)benzamide (31)
6-(4-cyclopentylpiperazin-1-yl)-N-(4-fluorobenzyl)-9-(3-(pyrrolidin-1-yl)-
propyl)-9H-purin-2-amine (32)
6-(4-ethylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-2-amine
(33)
6-(4-ethylpiperazin-1-yl)-N-(4-methoxybenzyl)-9-(3-(pyrrolidin-1-yl)-
propyl)-9H-purin-2-amine (34)
4-((dimethylamino)methyl)-N-(6-(4-ethylpiperazin-1-yl)-9-(3-(pyrrolidin-1-
-yl)propyl)-9H-purin-2-yl)benzamide (35)
6-(4-ethylpiperazin-1-yl)-N-(4-fluorobenzyl)-9-(3-(pyrrolidin-1-yl)propyl-
)-9H-purin-2-amine (36)
(4-(2-Amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-6-yl)piperazin-1-
-yl)(cyclopentyl)methanone (37)
Cyclopentyl(4-(2-(4-(diethylamino)benzylamino)-9-(3-(4-ethylpiperazin-1-y-
l)propyl)-9H-purin-6-yl)piperazin-1-yl)methanone (38)
Cyclopentyl(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-(4-methoxybenzylamin-
o)-9H-purin-6-yl)piperazin-1-yl)methanone (39)
Cyclopentyl(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-fluorobenzyl)ami-
no)-9H-purin-6-yl)piperazin-1-yl)methanone (40). In another
embodiment of the invention wherein compounds may be in a free form
or in pharmaceutically acceptable form.
In another embodiment of the invention wherein compounds may be
useful in treating various auto-immune diseases where activation of
TLR9 plays a role. In another embodiment of the invention wherein
compounds may be useful in inhibiting TLR9 mediated
immune-stimulatory signalling comprising contacting a cell
expressing a TLR9 with effective amount of these compounds. In
another embodiment of the invention wherein compounds are prepared
by the process wherein the process steps comprising: (i) reacting
6-chloro-9H-purin-2-amine with ethyl piperazine or
1-(3-chloropropyl)-4-ethylpiperazine in presence of a base in a
solvent at reflux temperature for a period ranging between 3 to 4
hrs to obtain compound 2 or 17, (ii) reacting compound 2 with
1-bromo-3-chloro propane in presence of a base in DMSO, DMF at a
room temperature for a period ranging between 10 to 12 hr to obtain
compound of formula 3, (iii) reacting compound 3 with ethyl
piperazine or pyrrolidine in presence of a base DIPEA or Et.sub.3N
in a solvent to obtain compound 4 or compound 33 respectively. (iv)
reacting compound 4 as obtained in step (iii) with a compound
selected from a group consisting of aromatic aldehyde, acid, acid
chloride, dibromoalkane in presence of a base and a solvent at a
temperature range between 25 to 110.degree. C., for a period 3 to
24 hr to obtain compound 5 to 16, (v) reacting compound 1 or 17
with a compound phenyl (piperazine-1-yl) methanone,
1-cyclopentylpiperazine or cyclopentyl(piperazin-1-yl)methanone in
presence of a base selected form a group comprising of
Cs.sub.2CO.sub.3, K.sub.2CO.sub.3 solvent selected from THF,
dioxane, CH.sub.3CN at a temperature ranging between 80 to
100.degree. C. to obtain compound 18, 23, 28 or 37, (vi) reacting
compound 18 or 23 with a compound selected from a group consisting
of aldehyde, acid or dibromo alkane in a solvent and in the
presence of a base at a temperature ranging between 80 to
110.degree. C. for a period ranging between 12 to 24 hr to obtain
compound 19 to 22, 24 to 27, (vii) reacting compound 28 with a
compound 1-(3-chloropropyl)pyrrolidine to give compound 29, (viii)
reacting compound 29, 33 or 37 with a compound of aldehyde or an
acid in presence of a solvent and a base, at a temperature ranging
between 25 to 100.degree. C., for a period ranging between 3 hr to
24 hr to obtain compound 30 to 32, 34 to 36 and 37 to 40. In
another embodiment of the invention wherein base used in step (i)
and step (ii) is selected from a group consisting of
Cs.sub.2CO.sub.3, K.sub.2CO.sub.3. In yet another embodiment of the
invention wherein solvent used in step (i) is selected from a group
consisting of acetonitrile, THF, dioxane, DMF. In another
embodiment of the invention wherein where solvent in step (iii) is
selected from a group consisting of toluene, DCM. In one more
embodiment of the invention wherein aromatic aldehyde, acid, acid
chloride, dibromoalkane in step (iv), (vi) and (viii) is selected
from a group consisting of benzaldehyde, p-anisaldehyde,
4-fluorobenzaldehyde, 4-diethylamino benzaldehyde, benzoyl
chloride, 1-(Tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid,
benzenesulphonyl chloride, acetyl chloride,
2-(Isobutoxycarbonyl)amino)-3-phenyl propionic acid and
dibromopropane 4-[(Dimethyl amino) methyl] benzaldehyde, 1,
4-dibrobutane, 4-[(Dimethyl amino) methyl] benzoic acid. In another
embodiment of the invention wherein base and reducing agent in step
(iv), (vi) and step (viii) is selected from group consisting of
pyridine, sodium hydride, sodium triacetoxy borohydride, sodium
cyanoborohydride, sodium borohydride. In another embodiment of the
invention wherein solvent in step (iv), (vi) and (viii) is selected
from group consisting of toluene, dichloromethane, THF, DMF.
Further embodiment of the invention wherein the compounds can be
used for the preparation of pharmaceutical composition comprising a
compound as claimed in claim (I) optionally along with
pharmaceutically-acceptable excipients. In another embodiment
according to this aspect of the invention, a screening method is
provided to screen compounds of general formula I for testing TLR9
antagonism in primary human plasmacytoid dendritic cells (pDCs)
purified from human peripheral blood mononuclear cells.
In another embodiment according to this aspect of the invention, a
screening method of affecting TLR mediated signalling in response
to a TLR ligand is provided, which involves detecting TLR9
antagonism of effective amount of a compound of general Formula (I)
using a reporter cell line that reports nuclear factor kappa B
expression downstream of TLR9 signalling.
In another embodiment according to this aspect of the invention,
said compounds with formula (I) described by the present invention
affect immune response mediated through TLR9.
In another embodiment according to this aspect of the invention,
compounds of general formula I described by the present invention
inhibit immune stimulation via TLR9 antagonism.
In another embodiment according to this aspect of the invention,
compounds of general formula (I) is useful whenever it is desirable
to alter TLR9 mediated signalling in response to a suitable TLR
ligand or TLR signalling agonist.
In another embodiment according to this aspect of the invention, it
is believed that the said compounds with formula (I) can be useful
to inhibit an immune stimulatory nucleic acid associated response
in a subject.
In another embodiment according to this aspect of the invention, it
is believed that the said compounds with general formula (I) that
can modulate autoreactive inflammation in different autoimmune
diseases where aberrant TLR9 activation is implicated for such
diseases.
In another embodiment according to this aspect of the invention, it
is believed that the said compounds with general formula (I) can be
used in a number of clinical applications, including as
pharmaceutical agents and methods for treating conditions involving
unwanted immune activity due to TLR9 activation.
In another embodiment according to this aspect of the invention,
the said compounds with formula (I) is believed to affect TLR9
directly and thus affect TLR-bearing cells, such as
antigen-presenting cells (APCs).
BRIEF DESCRIPTION OF DRAWING
FIG. 1: Structural evolution of the compound with general formula
(I) along with respective TLR9-antagonistic activity. The figure
denotes percent interferon alpha production in response to
TLR9-agonist ODN2216 from human peripheral blood mononuclear cells
in the presence of different doses of the compound with general
formula (I) (0, 0.1, 1, 5, 10, 20 .mu.M). Each row represents a
single molecule with increasing antagonist concentrations from left
to right as shown in the figure. TLR9-antagonist activity of one
representative molecule belonging to each structural subset is
indicated.
FIG. 2: TLR9 inhibition in HEK-Blue-hTLR9 reporter cell line by
selected compounds with general formula (I). The graphs denote
dose-dependent inhibition of TLR9 activation in a HEK-Blue-hTLR9
reporter cell line in the presence of different doses of the
compound with general formula (I), which is represented in terms of
decrease in SEAP activity. Data shown are mean of triplicate
wells.+-.SD.
FIG. 3: TLR9 inhibition in pDCs by selected compounds with formula
(I). The graphs denote dose-dependent reduction in IFN-.alpha.
production in response to TLR9-agonist ODN2216 from human
plasmacytoid dendritic cells (pDC) in the presence of different
doses of the antagonist molecules. Each data is derived from two
donors. Average values are reported.
FIG. 4: Cytotoxicity based on MTT assay of the identified TLR9
antagonist molecules with general formula (I). HepG2 and SW480
cells were cultured in presence of different concentrations (0.1,
0.5, 1, 10, 20 and 100 .mu.M) of different compound with general
formula (I) for 24 hrs. At 24 hrs MTT assay was performed as
described in the text. Respective absorbance at 570 nm is
represented. Each line represents a specific small molecule as
denoted in the legend.
Table 1 depicts overall structure of the compounds with general
formula (I) composition of the Invention
Table 2 depicts IC50 values of the compounds with general formula
(I) composition of the Invention.
ABBREVIATIONS
BnBr Benzylbromide
DMF N,N-dimethylformamide
AcOH Acetic acid
CDI 1,1'-Carbonyldiimidazole
POCl.sub.3 phosphorous oxychloride
DIPEA N,N-Diisopropylethylamine
DCM Dichloromethane
TFA Trifluoroacetic acid
DMSO Dimethyl sulfoxide
Boc Tert butyl carbamate
THF Tetrahydrofuran
HATU
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-
-oxide hexafluorophosphate
DETAILED DESCRIPTION OF THE INVENTION
The Present Invention is Described Here in Detail for Making and
Using the Compounds of Formula I
Compound 2 was prepared by using 6-chloro-9H-purin-2-amine and
ethylpiperazine as starting materials and potassium carbonate as a
base, in acetonitrile at reflux for 3-4 h. Compound 2 was treated
with 1-bromo-3-chloro propane in the presence of K.sub.2CO.sub.3 as
a base, in DMF at room temperature for 12 h to give compound 3.
Compound 3 was taken in a seal tube treated with the
ethylpiperazine and potassium carbonate as a base, in DMSO at
100.degree. C. for 24 hours the residue was separated by the column
chromatography to give compound 4. Subsequently, compound 4 was
treated with the 4-methoxybenzaldehyde under refluxing condition in
presence of the molecular sieves in toluene for 24 hours,
thereafter toluene was evaporated and sodium triacetoxyborohydride
was added and the reaction mixture was stirred for two hour,
residue was separated by column chromatography to get compound 5.
Compound 4 was similarly treated with 4-fluorobenzaldehyde under
refluxing condition in presence of molecular sieves in toluene for
24 hours. Subsequently sodium triacetoxyborohydride was introduced
and the reaction stirred for one hour at room temperature, the
residue was separated by the column chromatography to get the
compound 6. Compound 4 was treated with benzaldehyde and
4-diethylamino benzaldehyde separately in toluene at reflux for 12
h. After cooling sodium acetoxyborohydride was added and stirred
for 12 h at room temperature. The residue was purified by using
column chromatography to give compound 8 and 7. Compound 4 was
treated with 1,4-dibromobutane in DMF, NaH used as a base at
80.degree. C. and stirred for 24 hours, residue was separated by
the column chromatograph, to get compound 9. Compound 4 was treated
with benzoyl chloride in the presence of pyridine in DCM for 10 h
at room temperature and purified by column chromatography to
provide compound 10. 1-(tert-butoxycarbonyl)
Pyrrolidine-2-carboxylic acid was treated with oxalyl chloride and
catalytic amount of DMF and pyridine in DCM to produce acid
chloride in situ and compound 4 was added to this mixture to
undergo amide coupling to give compound 11 after column
chromatography. Compound 1 was treated with trifluoroacetic acid in
DCM at 0.degree. C. for 30 mins and quenched with ammonia and
purified by column chromatography to give compound 12. Compound 13
was prepared by treating compound 4 with benzene sulfonyl chloride
in presence of pyridine in DCM and DMAP was added at catalytic
amount and stirred for 10 h and the residue was purified by column
chromatography. Compound 4 was undergone amide coupling with acetyl
chloride, in presence of pyridine in DCM. The residue was purified
by using column chromatography to give compound 14.
2-(Isobutoxycarbonyl) amino)-3-phenylpropanoic acid was treated
with oxalyl chloride and catalytic amount of DMF and pyridine in
DCM to produce acid chloride in situ and compound 4 was added to
this mixture to undergo amide coupling to give compound 15 after
column chromatography purification. Compound 16 was synthesised by
treating compound 4 with 4-((dimethylamino)methyl)benzoic acid and
POCl.sub.3 in pyridine. The residue was purified by column
chromatography to give compound 16. Compound 17 was prepared by
treating compound 1 with 1-(3-choloropropyl)-4-ethylpiperazine in
presence of potassium carbonate as a base in DMF at 120.degree. C.
for 24 hours. The residue was separated by column chromatography,
to give compound 17, which was treated with phenyl
(piperazin-1-yl)methanone and potassium carbonate under refluxing
condition, in acetonitrile for 12 hours at 100.degree. C. to give
compound 18. Thereafter, compound 18 was treated with 4-diethyl
aminobenzaldehyde in toluene under refluxing condition for 24
hours. The solvent was evaporated and sodium triacetoxyborohydride
was introduced and the reaction mixture stirred for 2 hours to give
the compound 19. Compound 18 was also treated with 1, 4-dibrobutane
in DMF, at 60.degree. C. for 12 hours; residue is separated by
column chromatography to get the compound 20. Compound 17 was
treated with the 1-cyclopentenyl-4-ethylpiperazine, potassium
carbonate as a base, in acetonitrile at 100.degree. C. for 12
hours; residue was separated by the column chromatography to give
compound 23. Compound 23 was treated with 4-diethyl amino
benzaldehyde in toluene at 110.degree. C. for 24 hours and
subsequently sodium triacetoxyborohydride was added. The residue
was separated by column chromatography to get the compound 24.
Compound 23 was treated with 4-fluorobenzaldehyde in toluene at
110.degree. C. for 24 hours and subsequently sodium
triacetoxyborohydride was added and stirred at room temperature for
2 hours. The residue was purified by column chromatography to give
compound 25. Compound 26 was prepared by treating compound 23 with
4-methoxybenzaldehyde under refluxing condition in presence of the
molecular sieves in toluene for 24 hours. Thereafter toluene was
evaporated and sodium triacetoxyborohydride was added and the
reaction mixture was stirred for two hours in DCE. The residue was
purified by column chromatography to give compound 26. Compound 27
was synthesised by treating compound 23 with
4-((dimethylamino)methyl)benzoic acid and POCl.sub.3 in pyridine.
The residue was purified by column chromatography to give compound
27. Compound 28 was prepared by treating 6-chloro-9H-purin-2-amine
with 1-cyclopentylpiperazine and potassium carbonate as a base in
acetonitrile at 100.degree. C. for 12 hours.
1-(3-chloropropyl)pyrrolidine was added to a stirred suspension of
compound 28 and potassium carbonate in dry DMF and the reaction
mixture was heated for 12 hours at 80.degree. C. to give compound
29. Compound 30 was synthesised by treating compound 29 with
4-methoxybenzaldehyde under refluxing condition in presence of the
molecular sieves in toluene for 24 hours. Then toluene was
evaporated and sodium triacetoxyborohydride was added and the
reaction mixture was stirred for two hours in dichloroethane. The
residue was purified by column chromatography to obtain compound
30. Compound 31 was prepared by the reaction of compound 29 and
4-((dimethylamino)methyl)benzoic acid and POCl.sub.3 in pyridine.
The residue was purified by column chromatography to obtain
compound 31. Compound 29 was heated with 4-fluorobenzaldehyde in
toluene at 110.degree. C. for 24 hours and subsequently sodium
triacetoxyborohydride was added and stirred at room temperature for
2 hours. The residue was purified by column chromatography to get
compound 32. Compound 33 was synthesised by heating compound 3 with
pyrrolidine and triethylamine as base in DMSO at 110.degree. C. for
24 hours. Compound 34 was prepared by treating compound 33 with
4-methoxybenzaldehyde under refluxing condition in presence of the
molecular sieves in toluene for 24 hours. Then toluene was
evaporated and sodium triacetoxyborohydride was added and the
reaction mixture was stirred for two hours in dichloroethane. The
residue was purified by column chromatography to obtain compound
34. Compound 35 was synthesised by reacting compound 33 with
4-((dimethylamino)methyl)benzoic acid and POCl.sub.3 in pyridine.
The residue was purified by column chromatography to give compound
35. Compound 36 was prepared by reacting compound 33 with
4-fluorobenzaldehyde in toluene at 110.degree. C. for 24 hours and
subsequently sodium triacetoxyborohydride was added and stirred at
room temperature for 2 hours. The residue was purified by column
chromatography to obtain compound 36. Compound 17 was heated with
cyclopentyl(piperazin-1-yl)methanone and potassium carbonate in
acetonitrile for 12 hours at 100.degree. C. to give compound 37.
Compound 38 was synthesised by reacting compound 37 with
4-(diethylamino)benzaldehyde in toluene at refluxing condition for
24 hours. Toluene was evaporated and sodium triacetoxyborohydride
was added in dichloroethane. The residue was purified by column
chromatography to obtain compound 38. Compound 39 was prepared by
reacting 4-methoxybenzaldehyde under refluxing condition in
presence of the molecular sieves in toluene for 24 hours. Toluene
was evaporated and sodium triacetoxyborohydride was added and the
reaction mixture was stirred for two hours in dichloroethane. The
residue was purified by column chromatography to obtain compound
39. Compound 40 was synthesised by reacting with compound and
4-((dimethylamino)methyl)benzoic acid and POCl.sub.3 in pyridine.
The residue was purified by column chromatography to obtain
compound 40.
A screening method was used for evaluating TLR 9 antagonistic
activities of the synthesized compounds of general formula (I) by a
medium throughput biological assay based on TLR 9 activation in
primary human immune cells. The bona fide ligands used for TLR9
activation are Type A and type B unmethylated cytosine-guanine rich
DNA oligonucleotides (CpG oligonucleotides).
Type I interferons (e.g. IFN-alpha) are released on activation of
TLR9 by CpG oligonucleotides. The synthesized compounds of general
formula I was able to alter the release of type I interferons (e.g.
IFN-alpha).
The principle of the screening assay was designed based on the
production of Type I interferons (IFN-alpha) from human PBMCs,
which results almost exclusively from TLR9 triggering on the PDCs
by type A CpG oligonucleotides (CpGA). PBMCs were isolated from
venous blood collected from healthy donors using density gradient
centrifugation. The synthesized compounds of general formula (I)
having TLR9 antagonistic activity inhibited IFN-alpha production in
this screening assay (FIG. 1).
A screening method is provided using a HEK-Blue-hTLR9 Secreted
Alkaline Phosphatase (SEAP) reporter assay for the synthesized
compounds of general Formula (I) with TLR9 antagonism. The method
involves detecting TLR9 antagonistic activity for the synthesized
compounds of general Formula (I) by inhibiting TLR9-mediated NF-kB
activation in a dose-dependent manner (FIG. 2).
A biological assay based screening method is provided for compounds
of general formula (I) for testing TLR9 antagonism in primary human
plasmacytoid dendritic cells (pDCs) purified from human peripheral
blood mononuclear cells. The assay is based on toll-like receptor 9
activation in pDC (FIG. 3).
MTT assay for assessing cell viability is used to screen for
cytotoxicity for the synthesized compounds of general Formula (I).
In the cytotoxicity assay PBMC, HepG2 (a hepatic epithelial cell
line) and SW480 (an intestinal mucosal epithelial cell line) cells
were used. The synthesized compounds of general Formula I did not
show any considerable cytotoxicity at concentrations below 100 uM
on this assay (FIG. 4).
EXPERIMENTAL DETAILS
The following examples are intended for illustrative purposes only
and are not to be construed as being limitations for the invention
thereon in any manner. Temperatures are given in degree Celsius.
The structure of final products, intermediates and starting
materials is confirmed by standard analytical methods, e.g.
spectroscopic characterization, e.g., MS, NMR. Abbreviations used
are those conventional in the art.
All starting materials, reagents, catalysts, building blocks,
acids, bases, dehydrating agent and solvents utilized to synthesize
the compounds of the present invention are either commercially
available or can be produced by known organic synthesis methods in
the art.
##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011##
##STR00012##
##STR00013## ##STR00014## ##STR00015##
##STR00016## ##STR00017## ##STR00018##
##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023##
##STR00024## ##STR00025##
Following Examples are Given by Way of Illustration and should not
be Construed the Scope of the Invention
Example 1
Synthesis of 6-(4-ethylpiperazine-1-yl)-9H-purin-2-amine (2)
1-Ethylpiperazine (1.01 g, 8.84 mmol) was added to a stirred
suspension of 6-chloro-9H-purin-2-amine 1 (1 g, 5.89 mmol) and
potassium carbonate (0.8 g, 5.89 mmol) in dry acetonitrile. The
mixture was heated at reflux for 3-4 hours. acetonitrile was
removed under vacuum, the residue then washed with water, followed
by filtration and dried to obtain compound 2 as a light brown solid
(1.3 g, 89.6%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.61
(s, 1H), 4.22-4.31 (m, 7H), 2.56-2.63 (m, 4H), 2.50 (q, J=7.1 Hz,
H), 1.15 (t, J=7.2 Hz, 3H); ESI-MS m/z 248.20 (M+H).
Synthesis of
9-(3-chloropropyl)-6-(4-ethylpiperazine-1-yl)-9H-purin-2-amine
(3)
1-Bromo-3-chloropropane (2.4 mL, 24.26 mmol) was added to a stirred
suspension of compound 2 (2 g, 8.08 mmol) and potassium carbonate
(1.1 g, 8.08 mmol) in dry DMF and the reaction mixture was stirred
for 12 hours at room temperature. Water was added to the reaction
mixture. The aqueous solution was extracted with chloroform and the
organic layer was dried over Na.sub.2SO.sub.4 and evaporated under
vacuum. The residue was purified by silica gel column
chromatography eluting with 5% methanol in chloroform, to provide
compound 3 (1.65 g, 63%) as colorless liquid. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. ppm 7.50 (s, 1H), 4.61-4.71 (m, 2H), 4.18-4.36
(m, 6H), 3.50 (t, J=6.1 Hz, 2H), 2.56 (t, J=1.00 Hz, 4H), 2.46 (q,
J=7.14 Hz, 2H), 2.31 (q, J=1.00 Hz, 2H), 1.13 (t, J=7.23 Hz, 3H);
ESI-MS m/z 324.43 (M+H).
Synthesis of
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-2--
amine (4)
A solution of compound 3 (1 g, 3.09 mmol) and triethylamine (1.29
mL, 9.28 mmol) in dry DMSO was taken in a seal tube.
1-ethylpiperazine (0.43 mL, 3.4 mmol) was added to the stirred
mixture. The mixture was heated at 100.degree. C. for 12 hours.
Water was added to the reaction mixture. The aqueous solution was
extracted with chloroform. The organic layer was dried over
Na.sub.2SO.sub.4 and evaporated under vacuum. The residue was
purified by silica gel column chromatography, to provide compound 4
(0.65 g, 53%) as brown color gummy liquid. .sup.1H NMR (600 MHz,
CDCl.sub.3) .delta. ppm 7.48 (s, 1H), 4.61 (s, 2H), 4.16-4.32 (m,
4H), 4.07 (t, J=6.75 Hz, 2H), 2.54 (t, J=5.03 Hz, 5H), 2.38-2.51
(m, 8H), 2.35 (t, J=1.00 Hz, 1H), 2.30 (t, J=6.90 Hz, 2H), 2.20 (m,
2H), 1.97 (q, J=6.84 Hz, 2H), 1.11 (t, J=7.19 Hz, 3H), 1.07 (t,
J=7.23 Hz, 3H); ESI-MS m/z 402.47 (M+H).
Synthesis of
6-(4-ethylpiperazin-1-yl)-9-(-3-ethylpiperazin-1-yl)propyl)-N-(4-methoxyb-
enzyl) 9H-purin-2-amine (5)
Compound 4 (0.3 g, 0.74 mmol) and 4-methoxybenzaldehyde (0.10 mL,
0.89 mmol) were dissolved in toluene (5 mL). One pinch of molecular
sieves (3A powder) was added and the reaction stirred at
110.degree. C. under N.sub.2 atmosphere for 24 hours. Thereafter
toluene was evaporated and sodium triacetoxyborohydride (0.3 g,
1.48 mmoL) was added. The reaction was stirred at room temperature
for 1-2 hours. Reaction mixture was neutralized with NaHCO.sub.3
solution. Organic part was extracted with CHCl.sub.3 system. Column
chromatography was done by using CHCl.sub.3/CH.sub.3OH system to
get pure product of 5 (0.06 g, 45%) as a red liquid. .sup.1HNMR
(300 MHz, CDCl.sub.3) .delta. ppm 7.27 (s, 3H), 6.50 (d, J=9 Hz,
2H), 4.97 (s, 1H), 4.54 (d, J=6 Hz, 2H), 4.24 (s, 3H), 4.07 (d, J=6
Hz, 2H), 3.79 (s, 3H), 2.54 (s, 5H), 2.42 (t, J=6 Hz, 5H), 2.30 (d,
J=6 Hz, 3H), 1.98 (t, J=6 Hz, 2H), 1.13 (s, 1H), 1.07 (d, J=6 Hz,
6H); ESI-MS m/z 522.60 (M+H).
Synthesis of
6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4-fluoro-
benzyl)-9H-purin-2-amine (6)
Reaction of Compound 4 (0.3 g, 0.74 mmol) and 4-fluorobenzaldehyde
(0.23 g, 0.88 mmol). Column chromatography was done by using
CHCl.sub.3/MeOH system to get pure product of 6 (0.05 g, yield 30%)
as a gummy liquid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm
7.32 (s, 2H), 6.98 (m, 2H), 4.57 (d, J=6 Hz, 1H), 4.22 (s, 3H),
4.06 (d, J=6 Hz, 2H), 2.40-2.54 (m, 11H), 2.25 (d, J=9 Hz, 2H),
1.94-199 (m, 3H), 1.25 (s, 2H), 1.08 (t, J=9 Hz, 5H); ESI-MS m/z
510.45 (M+H).
Synthesis of
N-(4-(diethylamino)benzyl)-6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperaz-
in-1-yl)propyl)-9H-purin-2-amine (7)
Reaction of compound 4 (0.18 g, 0.44 mmol) and 4-diethylamino
benzaldehyde (0.137 mL, 1.34 mmol). The residue was purified by
silica gel column chromatography, to produce compound 7 (0.11 g,
50%). .sup.1H NMR (300 MHz, CDCL.sub.3) .parallel. ppm 1.10-1.16
(m, 12H) 1.99 (t, J=6.6 Hz, 2H) 2.32 (br. s., 2H), 2.40-2.49 (m,
10H), 2.54 (d, J=4.52 Hz, 4H), 3.33 (d, J=6.78 Hz, 4H) 4.09 (t,
J=6.6 Hz, 2H), 4.24 (br. s., 4H), 4.48 (d, J=5.65 Hz, 2H), 4.91 (t,
J=5.65 Hz, 1H) 6.63 (d, J=8.67 Hz, 2H), 7.22 (d, J=8.67 Hz, 2H),
7.46 (s, 1H); ESI-MS m/z 563.69 (M+H).
Synthesis of
N-benzyl-6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purin-2-amine (8)
Reaction of compound 4 (0.18 g, 0.44 mmol) and benzaldehyde (0.137
mL, 1.34 mmol). The residue was purified by silica gel column
chromatography, to produce compound 8 (0.11 g, 50%). .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 7.50 (s, 1H), 7.27-7.44 (m, 5H),
5.07 (t, J=1.00 Hz, 1H), 4.66 (d, J=6.03 Hz, 2H), 4.19-4.38 (m,
4H), 4.12 (t, J=6.8 Hz, 2H), 2.55-2.60 (m, 6H), 2.41-2.52 (m, 10H),
2.32-2.37 (m, 2H), 2.02 (d, J=6.78 Hz, 2H), 1.14 (m, 6H); ESI-MS
m/z 492.38 (M+H).
Example 2
Synthesis of
6-(4-ethylpiperazin-1-yl)-9(3-(4-ethylpiperazin-1-yl)propyl-2-(pyrrolidin-
-1-yl)-9H-purine (9)
Compound 4 (0.23 g, 0.57 mmol) was dissolved in DMF (5 mL), cooled
to 0.degree. C. and NaH (0.12 g, 0.85 mmol) and 1,4-dibromobutane
(1.5 mL, 1.7 mmol) were added. The reaction was stirred under the
N.sub.2 atmosphere condition at 80.degree. C. for 24 hours. Water
was added to the reaction mixture and the organic layer was
extracted with CHCl.sub.3. Column chromatography was done by using
CHCl.sub.3 system to get the compound 9 (0.04 g, yield 30%) as a
gummy liquid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.41
(s, 1H), 4.24 (s, 5H), 4.07 (t, J=6 Hz, 3H), 3.5 (t, J=6 Hz, 5H),
2.42-2.46 (m, 11H), 2.30 (d, J=9 Hz, 5H), 1.89-1.94 (m, 7H),
1.06-1.13 (m, 8H). ESI-MS m/z 456.48 (M+H).
Example 3
Synthesis of
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)benzamide (10)
Benzoyl chloride (0.2 mL, 1.49 mmol) was added to a stirred
solution of compound 4 (0.2 g, 0.498 mmol) and pyridine (0.24 mL,
2.99 mmol) in DCM. The mixture was stirred at room temperature for
10 hours. Solvent was removed under vacuum, the residue then washed
with water. The aqueous solution was extracted with chloroform,
washed with NaHCO.sub.3, dried over Na.sub.2SO.sub.4 and evaporated
under vacuum. The residue was purified by silica gel column
chromatography to provide compound 10 (0.15 g, 60%). .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 8.31 (s, 1H), 7.91 (d, J=7.1 Hz,
2H), 7.69 (s, 1H), 7.42-7.59 (m, 3H), 4.10-4.52 (m, 6H), 2.39-2.62
(m, 16H), 2.34 (t, J=6.68 Hz, 2H), 2.07 (t, J=1.00 Hz, 2H),
1.06-1.19 (m, 6H); ESI-MS m/z 506.52 (M+H).
Example 4
Synthesis of tert-butyl
2-((6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-puri-
n-2-yl) carbamoyl) pyrrolidine-1-carboxylate (11)
Oxalyl chloride (0.21 mL, 2.49 mmol) was added to DCM with
catalytic amount of DMF at 0.degree. C. Pyridine (0.24 mL, 2.99
mmol) was added to the reaction mixture and stirred for 15 mins.
1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (0.32 g, 1.3
mmol) was introduced and stirred for 25 mins. Compound 4 (0.2 g,
0.49 mmol) was dissolved in dry DCM and introduced into the
reaction mixture, stirred for 1 h. Water was added to the reaction
mixture. The aqueous solution was extracted with CHCl.sub.3. The
organic layer was washed with NaHCO.sub.3, dried over
Na.sub.2SO.sub.4 and evaporated under vacuum. The residue was
purified by silica gel column chromatography, to give compound 11
(0.15 g, 51%). .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. ppm 7.65
(s, 1H), 4.23-4.71 (m, 4H), 4.15-4.20 (m, 2H), 3.45-3.64 (m, 2H),
2.42-2.67 (m, 14H), 2.31 (t, J=6.57 Hz, 3H), 2.15-2.27 (m, 2H),
1.96-2.08 (m, 5H), 1.91 (dd, J=10.45, 4.29 Hz, 2H), 1.43-1.53 (m,
9H), 1.07-1.15 (m, 6H); ESI-MS m/z: 599.66 (M+H).
Synthesis of
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl) pyrrolidine-2-carboxamide (12)
Trifluoroacetic acid (0.1 mL, 1.3 mmol) was added to a solution of
compound 11 (0.08 g, 0.13 mmol) in DCM at 0.degree. C., stirred for
30 mins. The reaction was quenched using ammonia, DCM was removed
under vacuum, the residue then dissolved in CHCl.sub.3 (20 mL), and
the organic layer was washed with water, dried and concentrated.
The residue was purified by silica gel column chromatography, to
produce compound 12 (0.06 g, 91%). .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. ppm 9.85-10.14 (m, 1H), 7.69 (s, 1H), 4.27-4.49
(m, 4H), 4.23 (t, J=6.8 Hz, 2H), 3.91-4.00 (m, 1H) 3.06-3.15 (m,
2H), 2.58-2.64 (m, 5H), 2.48 (m, 10H), 2.35 (t, J=6.78 Hz, 2H),
2.19-2.30 (m, 2H), 2.16 (m, J=6.40 Hz, 2H), 2.09 (t, J=6.80 Hz,
2H), 1.75-1.88 (m, 2H), 1.14 (m, 6H); ESI-MS m/z 499.55 (M+H).
Example 5
Synthesis of
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)
propyl)-9H-purin-2-yl)benzenesulfonamide (13)
Benzenesulfonyl chloride (0.2 g, 1.22 mmol) was added to a stirred
solution of compound 4 (0.15 g, 0.37 mmol) and pyridine (0.18 mL,
2.24 mmol) in DCM. DMAP was added at catalytic amount. The mixture
was stirred at room temperature for 10 h. Solvent was removed under
vacuum, the residue then washed with water. The aqueous solution
was extracted with chloroform, washed with NaHCO.sub.3, dried over
Na.sub.2SO.sub.4 and evaporated under vacuum. The residue was
purified by silica gel column chromatography, to provide compound
13 (0.1 g, 50%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 8.09
(d, J=7.16 Hz, 2H), 7.40-7.65 (m, 4H), 4.08 (m, J=6.60, 6.60 Hz,
6H), 3.02 (s, 1H), 2.36-2.64 (m, 16H), 2.29 (t, J=1.00 Hz, 2H),
1.94 (t, J=1.00 Hz, 2H), 1.02-1.16 (m, 6H); ESI-MS m/z 542.48
(M+H).
Example 6
Synthesis of
N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-
-2-yl)acetamide (14)
Acetyl chloride (0.1 mL, 1.49 mmol) was added to a stirred solution
of compound 4 (0.2 g, 0.49 mmol) and pyridine (0.24 mL, 2.99 mmol)
in DCM. The mixture was stirred at room temperature for 10 hours.
Solvent was removed under vacuum, the residue then washed with
water. The aqueous solution was extracted with chloroform. The
organic layer was dried over Na.sub.2SO.sub.4 and evaporated under
vacuum. The residue was purified by silica gel column
chromatography, to give compound 14 (0.11 g, 82%). .sup.1H NMR (300
MHz, DMSO) .delta. ppm 7.75 (s, 1H), 7.64 (s, 1H), 4.10-4.48 (m,
6H), 2.53-2.57 (m, 6H), 2.36-2.50 (m, 10H), 2.29 (t, J=6.78 Hz,
3H), 1.93-2.05 (m, 4H), 1.06-1.16 (m, 6H); ESI-MS m/z 444.43
(M+H).
Example 7
Synthesis of iso-butyl
(1-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-puri-
n-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (15)
A solution of compound 4 (0.25 g, 0.62 mmol) and
2-((tert-butoxycarbonyl)amino)-3-phenylpropanoic acid (0.16 g, 0.62
mmol) in pyridine was added POCl.sub.3 (0.06 mL, 0.68 mmol) at
-10.degree. C. and stirred for 2 hours. The reaction mixture was
quenched with ammonia until it becomes neutral pH. Ice was added to
the mixture. The aqueous layer was extracted with chloroform, dried
over Na.sub.2SO.sub.4 and evaporated under vacuum. The residue was
purified by silica gel column chromatography to provide compound 15
(0.21 g, 53%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm
7.89-7.98 (m, 1H), 7.65 (s, 1H), 7.19-7.28 (m, 5H), 5.15-5.29 (m,
1H), 4.08-4.44 (m, 6H), 3.25 (dd, J=1.00 Hz, 1H), 3.04-3.16 (m,
1H), 2.37-2.60 (m, 15H), 2.29 (t, J=1.00 Hz, 2H), 1.93-2.08 (m,
4H), 1.33-1.47 (m, 9H), 1.11 (m, 6H); ESI-MS m/z 649.83 (M+H).
Synthesis of
4-((dimethylamino)methyl)-N-(6-(4-ethylpiperazin-1-yl)-9-(3-(4-ethylpiper-
azin-1-yl)propyl)-9H-purin-2-yl)benzamide (16)
Reaction of Compound 4 (0.2 g, 0.5 mmol) and
4-((dimethylamino)methyl)benzoic acid (0.13 g, 0.6 mmol) was done
according to procedure B. Then column chromatography was done by
using CHCl.sub.3/MeOH system to get the pure product 16 (yield
53%). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 8.06 (s,
--NH), 7.86 (d, J=8.4 Hz, 2H), 7.70 (s, 1H), 7.40 (d, J=7.8 Hz,
2H), 4.12 (m, 6H), 3.97 (t, J=6.0 Hz, 2H), 3.16-3.09 (m, 2H), 2.72
(s, 6H), 2.47-2.41 (m, 8H), 2.40-2.37 (m, 4H), 2.27-2.25 (m, 4H),
1.96-1.84 (m, 2H), 1.03 (t, J=6.3 Hz, 6H). ESI-MS m/z 563.32
(M+H).
Example 8
Synthesis of
6-chloro-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-2-amine
(17)
Compound 1 (2 g, 0.01 mmol) was dissolved in DMF (5 mL), and
stirred for 2-3 hours under the N2 atmosphere condition at the
120.degree. C. get the clear solution. Potassium carbonate and (1.6
g, 0.1 mmol), (3-chloropropyl)-4-ethylpiperazine) were added to the
reaction mixture and stirred for 12 hours. Water was added to the
reaction and organic layer was separated with CHCl.sub.3. Column
chromatography was done by using CHCl.sub.3/CH.sub.3OH system, to
get the compound 17 (1.2 gm, 50% yield) as a thick gummy liquid.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.81 (s, 1H), 5.21
(s, 2H), 4.14-4.19 (t, 2H), 2.43 (t, 10H), 2.29 (d, J=6 Hz, 3H),
2.2 (t, J=6 Hz, 3H), 1.08 (t, 4H); ESI-MS m/z 324.26 (M+H).
Synthesis of
(4-(2-amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-6-yl)piperazin-1-
-yl)(phenyl)methanone (18)
Compound 17 was dissolved in acetonitrile (5 mL), potassium
carbonate (1.2 g, 0.03 mmol) and phenyl(piperazine-1-yl) methanone
(0.84 g, 0.03 mmol) were added. The reaction was stirred for 2
hours under N.sub.2 atmosphere condition at 100.degree. C.
Acetonitrile was evaporated under the vacuum, then water was added
to the reaction mixture to get precipitate which was filter off to
get precipitate (0.8 g, yield 70%) of product 18. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. ppm 7.49 (s, 1H), 7.42 (d, J=9 Hz, 4H),
4.63 (s, 2H), 4.26 (s, 3H), 4.09 (t, J=6 Hz, 2H), 3.89 (s, 2H),
3.54 (s, 2H), 2.42 (d, J=6 Hz, 8H), 2.28-2.40 (m, 3H), 1.85-2.03
(m, 3H), 1.08 (t, J=6 Hz, 3H). ESI-MS m/z 478.34 (M+H).
Synthesis of
4-(2-(4-(diethylamino)benzyl)amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purine-6-yl)piperazin-1-yl)(phenyl)methanone (19)
Compound 18 (0.2 g, 0.41 mmol) and 4-diethyl aminobenzaldehyde
(0.07 g, 0.41 mmol) was dissolved in toluene (5 mL). Molecular
sieves (3 .ANG. powder) was added and the reaction stirred at
110.degree. C. under N.sub.2 atmosphere for 12-16 hours. Thereafter
toluene was evaporated and sodium triacetoxyborohydride (0.18 g,
0.82 mmol) to the reaction mixture and the mixture was dissolved in
DCE (5 mL). Reaction mixture was kept in room temperature and
allowed to stir for 2-3 hours. After completion of the reaction, it
was neutralized with NaHCO.sub.3 solution. Organic part was
extracted with 20% CH.sub.3OH/CHCl.sub.3 system. Column
Chromatography was done by using CHCl.sub.3 system to get pure
product of 19, (0.06 g, yield 52%) as a gummy liquid. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 7.47 (s, 6H), 7.19 (s, 2H), 6.63
(d, J=9 Hz, 2H), 4.96 (s, 1H), 4.45 (d, J=9 Hz, 2H), 4.21-4.27 (m,
3H), 4.07-4.12 (m, 2H), 3.87 (s, 2H), 3.29-3.36 (m, 4H), 2.44-2.49
(m, 9H), 2.32 (d, J=6 Hz, 4H), 1.98 (d, J=6 Hz, 3H), 1.25 (s, 2H),
1.10-1.16 (m, 9H). ESI-MS m/z 639.74 (M+H).
Synthesis of
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-(pyrrolidin-1-yl)-9H-purin-6-yl-
)piperazin-1-yl)(phenyl)methanone (20)
Compound 18 (0.25 g, 0.52 mmol) was dissolved in dry DMF (5 mL),
the reaction mixture was cooled to -10.degree. C. and NaH (0.02 g
1.4 mmol) was added. The reaction mixture was allowed to stir for
one hour at cold condition. Then 1,4-dibromo butane (0.06 g, 0.52
mmol) was added to it and allowed to stir for further 12 hours
under the N.sub.2 atmosphere at 60.degree. C. After completion of
the reaction, organic layer was extracted by using the
CHCl.sub.3/CH.sub.3OH system. Column chromatography was done by
using CHCl.sub.3/ system to get pure product 20 (0.02 g, yield 30%)
as a red colour gummy liquid. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. ppm 7.46 (s, 1H), 6.98 (t, J=9 Hz, 3H), 4.57 (d, J=6 Hz,
1H), 4.22 (s, 2H), 4.07 (s, 2H), 2.40-2.54 (m, 11H), 2.28 (d, J=6
Hz, 2H), 1.97 (d, J=9 Hz, 3H), 1.25 (s, 2H), 1.08 (t, J=9 Hz, 5H).
ESI-MS m/z 532.57 (M+H).
Synthesis of
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-methoxybenzyl)amino)-9H-pur-
in-6-yl)piperazin-1-yl)(phenyl)methanone (21)
Reaction of compound 18 (0.200 g, 0.42 mmol) and
4-methoxybenzaldehyde (0.137 mL, 1.34 mmol) was done as discussed
above for compound 5. The residue was purified by silica gel column
chromatography, to produce compound 21 (0.100 g, 66%). .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 7.44 (s, 1H), 7.42 (m, 5H), 7.27
(d, J=9.0 Hz, 2H), 6.83 (d, J=9.0 Hz, 2H), 5.25 (t, J=1.00 Hz, 1H),
4.52 (d, J=6.03 Hz, 2H), 4.20 (m, 4H), 4.07 (t, J=6.78 Hz, 2H),
3.85 (m, 2H), 3.78 (s, 3H), 3.51 (m, 2H), 2.50-2.57 (m, 10H), 2.34
(t, J=6.78 Hz, 2H), 1.96-1.98 (m, 2H), 1.12 (t, J=6.01, 3H); ESI-MS
m/z 598.14 (M+H.sup.+).
Synthesis of
(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-fluorobenzyl)amino)-9H-puri-
n-6-yl)piperazin-1-yl)(phenyl)methanone (22)
Reaction of compound 18 (0.200 g, 0.42 mmol) and
4-flurobenzaldehyde (0.06 mL, 1.34 mmol) was done as discussed
above for compound 5. The residue was purified by silica gel column
chromatography, to produce compound 22 (yield 63%). .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 7.43 (m, 6H), 7.31 (d, J=9.0 Hz,
2H), 6.99 (t, J=6.0 Hz, 2H), 5.29 (t, J=1.00 Hz, 1H), 4.55 (d,
J=6.03 Hz, 2H), 4.22 (m, 4H), 4.07 (t, J=6.78 Hz, 2H), 3.85 (m,
2H), 3.51 (m, 2H), 2.79-2.69 (m, 10H), 2.40 (t, J=6.78 Hz, 2H),
1.99 (t, J=6.0 Hz, 2H), 1.28 (t, J=6.01, 3H); ESI-MS m/z 586.05
(M+H).
Synthesis of 6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethyl
piperazin-1-yl)propyl)-9H-purin-2-amine (23)
Compound 17 (0.1 g, 0.36 mmol) was dissolved in acetonitrile (5 mL)
and potassium carbonate (0.04 g, 0.36 mmol) and
1-cyclopentylpiperazine (0.05 g, 0.37 mmol) were added. The
reaction was stirred under the N.sub.2 atmospheric condition at
100.degree. C. for 12 hours. Organic layer was extracted with
CHCl.sub.3 system and column chromatography was done by using
CH.sub.3OH and CHCl.sub.3 system to separate compound 23 (0.02 g,
yield 40%) as a brown liquid. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. ppm 7.46 (s, 1H), 4.71 (s, 2H), 4-4.10 (m, 5H), 2.24-2.56
(m, 18H), 1.93 (t, J=6 Hz, 6H), 1-1.05 (t, J=6 Hz, 5H). ESI-MS m/z
442.45 (M+H).
Example 9
Synthesis of
6-(4-cyclopentylpiperazin-1-yl)-N-(4-diethylamino)benzyl)-9-(3-(4-ethylpi-
perazine-1-yl)propyl)-9H-purine-2-amine (24)
Compound 23 (0.25 g, 0.34 mmol) and 4-diethyl aminobenzaldehyde
(0.20 g, 0.60 mmol) was dissolved in toluene (5 mL). Molecular
sieves (3 .ANG. powder) was added and the reaction stirred at
110.degree. C. under N.sub.2 atmosphere for 12-16 hours. Thereafter
toluene was evaporated and sodium triacetoxyborohydride (0.2 g,
0.68 mmol) was added to the reaction mixture and stirred for 2-3
hours at room temperature. Reaction mixture was neutralized with
NaHCO.sub.3 solution. Organic part was extracted with CHCl.sub.3.
Column chromatography was done by using CHCl.sub.3:CH.sub.3OH
system to get pure product of 24 (yield 55%) as a gummy liquid.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.43 (s, 1H), 7.19
(d, J=9.0 Hz, 2H), 6.61 (d, J=9 Hz, 2H), 4.45 (d, J=3 Hz, 2H), 4.42
(s, 3H) 4.06 (t, J=6. Hz, 2H), 3.27-3.34 (m, 3H), 2.50-2.57 (m,
5H), 2.37-2.42 (m, 9H), 2.28 (d, J=6 Hz, 3H), 1.97 (t, J=6. Hz,
2H), 1.86 (s, 2H), 1.68 (d, J=6. Hz, 2H), 1.41-1.55 (m, 4H), 1.03
(m, 10H). ESI-MS m/z 603.64 (M+H).
Synthesis of
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4--
fluorobenzyl)-9H-purin-2-amine (25)
Reaction of compound 23 (0.20 g, 0.42 mmol) and 4-flurobenzaldehyde
(0.06 mL, 1.34 mmol) was done as discussed above for compound 5.
The residue was purified by silica gel column chromatography, to
produce compound 25 (yield 61%). .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. ppm 7.46 (s, 1H), 7.32 (dd, J=9.0 Hz, 2H), 6.98 (t, J=9.0
Hz, 2H), 5.02 (m, 1H), 4.57 (d, J=6.03 Hz, 2H), 4.22 (m, 4H), 4.07
(t, J=6.78 Hz, 2H), 2.59-2.56 (m, 4H), 2.52-2.37 (m, 10H), 2.30 (t,
J=6.78 Hz, 2H), 1.96 (m, 2H), 1.70 (m, 2H), 1.40-1.43 (m, 4H), 1.08
(t, J=6.01, 3H); ESI-MS m/z 450.28 (M+H).
Synthesis of
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-N-(4--
methoxybenzyl)-9H-purin-2-amine (26)
Reaction of Compound 23 (0.2 g, 0.45 mmol) and
4-methoxybenzaldehyde (0.066 mL, 0.54 mmol was done as discussed
above for compound 5. Then column chromatography was done by using
CHCl.sub.3/MeOH system to get the pure product 26 (yield 56%) as a
gummy solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.45 (s,
1H), 7.29 (d, J=8.7 Hz, 2H), 6.85 (d, J=8.7 Hz, 2H), 5.0 (t, J=5.7
Hz, 1H), 4.54 (d, J=6.0 Hz, 2H), 4.29 (m, 4H), 4.08 (t, J=6.6 Hz,
2H), 3.79 (s, 3H), 2.65 (m, 4H), 2.57-2.53 (m, 8H), 2.35 (t, J=6.6
Hz, 2H), 2.03-1.97 (m, 3H), 1.91 (m, 2H), 1.73 (m, 2H), 1.55 (m,
2H), 1.26 (m, 4H), 1.15 (t, J=7.2 Hz, 3H). ESI-MS m/z 562.34
(M+H).
Synthesis of
N-(6-(4-cyclopentylpiperazin-1-yl)-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-
-purin-2-yl)-4-((dimethylamino)methyl)benzamide (27)
A solution of compound 23 (0.10 g, 0.23 mmol) and
4-((dimethylamino)methyl)benzoic acid (0.048 g, 0.27 mmol) in
pyridine was added POCl.sub.3 (0.03 mL, 0.35 mmol) at 0.degree. C.
and stirred for 2 hours. The reaction mixture was poured into
crushed ice and neutralised with saturated Na.sub.2CO.sub.3
solution. The aqueous layer was extracted with chloroform, dried
over Na.sub.2SO.sub.4 and evaporated under vacuum. The residue was
purified by silica gel column chromatography to give compound 27
(yield 53%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 8.30 (s,
--NH), 7.87 (d, J=7.8 Hz, 2H), 7.67 (s, 1H), 7.43 (d, J=7.5 Hz,
2H), 4.22 (t, J=6.9 Hz, 1H), 3.50 (s, 2H), 2.64 (m, 8H), 2.55 (m,
8H), 2.37 (t, J=6.6 Hz, 2H), 2.27 (s, 6H), 2.06 (t, J=6.6 Hz, 2H),
1.9 (m, 2H), 1.72 (m, 2H), 1.59-1.44 (m, 4H), 1.25 (m, 4H), 1.15
(t, J=7.5 Hz, 3H). ESI-MS m/z 603.26 (M+H).
Example 10
Synthesis of 6-(4-cyclopentylpiperazin-1-yl)-9H-purin-2-amine
(28)
Compound 1 (0.5 g, 2.95 mmol) was dissolved in acetonitrile (8 mL)
and potassium carbonate (1.63 g, 11.8 mmol) and
1-cyclopentylpiperazine (0.91 g, 5.9 mmol) were added. The reaction
was stirred under the N.sub.2 atmospheric condition at 100.degree.
C. for 12 hours. Organic layer was extracted with CHCl.sub.3 system
and column chromatography was done by using CH.sub.3OH and
CHCl.sub.3 system to give compound 28 (yield 60%) as a off-white
solid. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm 7.66 (s,
1H), 5.72 (s, --NH.sub.2), 4.09 (m, 4H), 2.46 (t, J=4.2 Hz, 4H),
2.42-2.39 (m, 1H), 1.61-1.50 (m, 4H), 1.43-1.29 (m, 4H). ESI-MS m/z
288.14 (M+H).
Synthesis of
6-(4-cyclopentylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-2--
amine (29)
1-(3-chloropropyl)pyrrolidine (0.77 g, 5.22 mmol) was added to a
stirred suspension of compound 28 (1 g, 3.48 mmol) and potassium
carbonate (0.96 g, 6.96 mmol) in dry DMF and the reaction mixture
was heated for 12 hours at 80.degree. C. Water was added to the
reaction mixture. The aqueous solution was extracted with
chloroform and the organic layer was dried over Na.sub.2SO.sub.4
and evaporated under vacuum. The residue was purified by silica gel
column chromatography eluting with 5% methanol in chloroform, to
give compound 29 (yield 65%) as colorless liquid. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. ppm 7.49 (s, 1H), 4.63 (s, --NH.sub.2),
4.24 (m, 4H), 4.09 (t, J=6.9 Hz, 2H), 2.59 (t, J=5.1 Hz, 4H), 2.50
(m, 4H), 2.46-2.41 (m, 4H), 2.16 (s, 1H), 2.01 (m, 2H), 1.87-1.83
(m, 2H), 1.72-1.63 (m, 4H), 1.52-1.41 (m, 4H). ESI-MS m/z 399.12
(M+H).
Synthesis of
6-(4-cyclopentylpiperazin-1-yl)-N-(4-methoxybenzyl)-9-(3-(pyrrolidin-1-yl-
)propyl)-9H-purin-2-amine (30)
Reaction of Compound 29 (0.2 g, 0.50 mmol) and
4-methoxybenzaldehyde (0.07 mL, 0.60 mmol) was done as discussed
above for compound 5. Then column chromatography was done by using
CHCl.sub.3/MeOH system to get the pure product 30 (yield 59%).
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.47 (s, 1H), 7.29
(d, J=8.4 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 4.99 (t, J=5.7 Hz,
--NH), 4.54 (d, J=5.7 Hz, 2H), 4.24 (m, 4H), 4.11 (t, J=7.2 Hz,
2H), 3.79 (s, 3H), 2.61-2.58 (m, 8H), 2.50 (t, J=7.2 Hz, 4H),
2.12-2.04 (m, 2H), 1.88 (m, 3H), 1.81 (m, 4H), 1.71 (m, 2H),
1.61-1.53 (m, 2H). ESI-MS m/z 519.19 (M+H).
Synthesis of
N-(6-(4-cyclopentylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-
-2-yl)-4-((dimethylamino)methyl)benzamide (31)
Reaction of Compound 29 (0.1 g, 0.25 mmol) and
4-((dimethylamino)methyl)benzoic acid (0.065 g, 0.30 mmol) was done
as discussed above for compound 27. Then column chromatography was
done by using CHCl.sub.3/MeOH system to get the pure product 31
(yield 53%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 8.33 (s,
--NH), 7.87 (d, J=7.8 Hz, 2H), 7.67 (s, 1H), 7.42 (d, J=8.1 Hz,
2H), 4.23 (t, J=6.6 Hz, 2H), 3.48 (s, 2H), 2.62-2.48 (m, 12H), 2.25
(s, 6H), 2.12 (t, J=6.6 Hz, 1H), 1.61-1.52 (m, 2H), 1.47-1.41 (m,
2H), 1.33-1.28 (m, 4H), 0.87-0.82 (m, 8H). ESI-MS m/z 560.34
(M+H).
Synthesis of
6-(4-cyclopentylpiperazin-1-yl)-N-(4-fluorobenzyl)-9-(3-(pyrrolidin-1-yl)-
propyl)-9H-purin-2-amine (32)
Reaction of Compound 29 (0.1 g, 0.25 mmol) and 4-fluorobenzaldehyde
(0.03 mL, 0.3 mmol) was done as discussed above for compound 5.
Then column chromatography was done by using CHCl.sub.3/MeOH system
to get the pure product 32 (yield 52%). 1H NMR (300 MHz,
CDCl.sub.3) .delta. ppm 7.47 (s, 1H), 7.35-7.31 (m, 2H), 6.98 (t,
J=8.7 Hz, 2H), 5.07 (t, J=6.0 Hz, --NH), 4.57 (d, J=6.0 Hz, 2H),
4.23 (m, 4H), 4.09 (t, J=6.9 Hz, 2H), 2.59-2.45 (m, 12H), 2.04 (m,
1H), 1.73-1.68 (m, 2H), 1.59-1.54 (m, 2H), 1.25 (m, 4H), 0.87-0.85
(m, 4H). ESI-MS m/z 507.18 (M+H).
Example 11
Synthesis of
6-(4-ethylpiperazin-1-yl)-9-(3-(pyrrolidin-1-yl)propyl)-9H-purin-2-amine
(33)
A solution of compound 3 (1 g, 3.09 mmol) and triethylamine (1.29
mL, 9.28 mmol) in dry DMSO was taken in a seal tube. Pyrrolidine
(0.28 mL, 3.4 mmol) was added to the stirred mixture. The mixture
was heated at 100.degree. C. for 12 hours. Water was added to the
reaction mixture. The aqueous solution was extracted with
chloroform. The organic layer was dried over Na.sub.2SO.sub.4 and
evaporated under vacuum. The residue was purified by silica gel
column chromatography, to provide compound 33 (72%). .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. ppm 7.50 (s, 1H), 4.61 (s,
--NH.sub.2), 4.25 (m, 4H), 4.11 (t, J=6.9 Hz, 2H), 2.56-2.53 (m,
8H), 2.49-2.42 (m, 6H), 2.20 (m, 2H), 2.04 (q, J=6.9 Hz, 2H), 1.12
(t, J=7.2 Hz, 3H). ESI-MS m/z 359.11 (M+H).
Synthesis of
6-(4-ethylpiperazin-1-yl)-N-(4-methoxybenzyl)-9-(3-(pyrrolidin-1-yl)propy-
l)-9H-purin-2-amine (34)
Reaction of Compound 33 (0.08 g, 0.22 mmol) and
4-methoxybenzaldehyde (0.03 mL, 0.27 mmol) was done as discussed
above for compound 5. Then column chromatography was done by using
CHCl.sub.3/MeOH system to get the pure product 34 (58%). .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.47 (s, 1H), 7.29 (d, J=8.4
Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 4.98 (t, J=5.4 Hz, --NH), 4.54 (d,
J=6.0 Hz, 2H), 4.23 (m, 4H), 4.10 (t, J=6.9 Hz, 2H), 3.79 (s, 3H),
2.53-2.44 (m, 12H), 2.05-1.99 (m, 6H), 1.12 (t, J=7.2 Hz, 3H).
ESI-MS m/z 479.21 (M+H).
Synthesis of
4-((dimethylamino)methyl)-N-(6-(4-ethylpiperazin-1-yl)-9-(3-(pyrrolidin-1-
-yl)propyl)-9H-purin-2-yl)benzamide (35)
Reaction of Compound 33 (0.09 g, 0.25 mmol) and
4-((dimethylamino)methyl)benzoic acid (0.065 g, 0.30 mmol)) was
done as discussed above for compound 27. Then column chromatography
was done by using CHCl.sub.3/MeOH system to get the pure product 35
(56%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 8.33 (s,
--NH), 7.87 (d, J=8.1 Hz, 2H), 7.69 (s, 1H), 7.43 (d, J=8.1 Hz,
2H), 4.24 (t, J=6.9 Hz, 2H), 3.49 (s, 2H), 2.67 (m, 4H), 2.58 (t,
J=4.8 Hz, 4H), 2.47 (q, J=7.2 Hz, 2H), 2.26 (s, 6H), 2.15 (t, J=6.9
Hz, 2H), 2.01 (m, 4H), 1.85 (m, 2H), 1.25 (m, 4H), 1.13 (t, J=6.9
Hz, 3H). ESI-MS m/z 520.09 (M+H).
Synthesis of
6-(4-ethylpiperazin-1-yl)-N-(4-fluorobenzyl)-9-(3-(pyrrolidin-1-yl)propyl-
)-9H-purin-2-amine (36)
Reaction of Compound 33 (0.08 g, 0.22 mmol) and
4-fluorobenzaldehyde (0.03 mL, 0.3 mmol) was done as discussed
above for compound 5. Then column chromatography was done by using
CHCl.sub.3/MeOH system to get the pure product 36 (58%). .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.74 (s, 1H), 7.35-7.31 (m,
2H), 7.01 (t, J=8.7 Hz, 2H), 4.59 (br. s, 2H), 4.36 (t, J=5.7 Hz,
2H), 2.54 (m, 8H), 2.48-2.41 (m, 4H), 2.28-2.20 (m, 2H), 1.77 (m,
2H), 1.63 (m, 4H), 1.25 (m, 2H), 1.11 (t, J=6.9 Hz, 3H). ESI-MS m/z
467.15 (M+H).
Example 12
Synthesis of
(4-(2-amino-9-(3-(4-ethylpiperazin-1-yl)propyl)-9H-purin-6-yl)piperazin-1-
-yl)(cyclopentyl)methanone (37)
Compound 17 was dissolved in acetonitrile (5 mL), potassium
carbonate (1.2 g, 0.03 mmol) and
cyclopentyl(piperazine-1-yl)methanone (0.84 g, 0.03 mmol) were
added. The reaction was stirred for 2 hours under N.sub.2
atmosphere condition at 100.degree. C. acetonitrile was evaporated
under the vacuum, then water was added to the reaction mixture to
get precipitate which was filter off to get precipitate (yield 75%)
of product 37. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.48
(s, 1H), 5.09 (t, J=1.00 Hz, 1H), 4.57 (d, J=6.03 Hz, 2H),
4.18-4.25 (m, 4H), 4.11 (t, J=7.5 Hz, 2H), 3.74 (m, 2H), 3.64 (m,
2H), 2.91 (m, 1H), 2.79-2.69 (m, 10H), 2.34 (t, J=6.78 Hz, 2H),
2.00 (m, 4H), 1.98 (m, 2H), 1.75 (m, 4H), 1.59 (m, 2H), 1.16 (t,
J=6.01, 3H); ESI-MS m/z 590.30 (M+H).
Synthesis of
cyclopentyl(4-(2-((4-(diethylamino)benzyl)amino)-9-(3-(4-ethylpiperazin-1-
-yl)propyl)-9H-purin-6-yl)piperazin-1-yl)methanone (38)
Reaction of compound 37 (0.20 g, 0.41 mmol) and
4-(diethylamino)benzaldehyde (0.88 gm, 1.34 mmol) was done as
discussed above for compound 5. The residue was purified by silica
gel column chromatography, to produce compound 38 (68%). .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. ppm 7.45 (s, 1H), 7.20 (d, J=9.0
Hz, 2H), 6.63 (d, J=9.0 Hz, 2H), 5.09 (s, 1H), 4.47 (d, J=6.03 Hz,
2H), 4.17 (m, 4H), 4.10 (t, J=6.78 Hz, 2H), 3.73 (m, 2H), 3.62 (m,
2H), 3.34 (m, 4H), 2.91 (m, 1H), 2.60 (m, 8H), 2.33 (m, 2H), 2.01
(m, 6H), 1.84 (m, 4H), 1.14 (t, J=6.01, 9H); ESI-MS m/z 631.16
(M+H).
Synthesis of
cyclopentyl(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-methoxybenzyl)am-
ino)-9H-purin-6-yl)piperazin-1-yl)methanone (39)
Reaction of compound 37 (0.200 g, 0.41 mmol) and
4-methoxybenzaldehyde (0.05 mL, 1.34 mmol) was done as discussed
above for compound 5. The residue was purified by silica gel column
chromatography, to produce compound 39 (63%). .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. ppm 7.48 (s, 1H), 7.31 (d, J=9.0 Hz, 2H), 6.86
(d, J=9.0 Hz, 2H), 5.09 (t, J=1.00 Hz, 1H), 4.57 (d, J=6.03 Hz,
2H), 4.18-4.25 (m, 4H), 4.11 (t, J=7.5 Hz, 2H), 3.81 (s, 3H), 3.74
(m, 2H), 3.64 (m, 2H), 2.91 (m, 1H), 2.79-2.69 (m, 10H), 2.34 (t,
J=6.78 Hz, 2H), 2.00 (m, 4H), 1.98 (m, 2H), 1.75 (m, 4H), 1.59 (m,
2H), 1.16 (t, J=6.01, 3H); ESI-MS m/z 590.30 (M+H).
Synthesis of
cyclopentyl(4-(9-(3-(4-ethylpiperazin-1-yl)propyl)-2-((4-fluorobenzyl)ami-
no)-9H-purin-6-yl)piperazin-1-yl)methanone (40)
Reaction of compound 37 (0.200 g, 0.41 mmol) and
4-flurobenzaldehyde (0.05 mL, 1.34 mmol) was done as discussed
above for compound 5. The residue was purified by silica gel column
chromatography, to produce compound 40 (61%). .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. ppm 7.47 (s, 1H), 7.32 (d, J=9.0 Hz, 2H), 6.99
(t, J=6.0 Hz, 2H), 5.09 (t, J=1.00 Hz, 1H), 4.57 (d, J=6.03 Hz,
2H), 4.22-4.15 (m, 4H), 4.08 (t, J=6.78 Hz, 2H), 3.71 (m, 2H), 3.60
(m, 2H), 2.91 (m, 1H), 2.79-2.69 (m, 10H), 2.34 (t, J=6.78 Hz, 2H),
2.00 (m, 4H), 1.98 (m, 2H), 1.75 (m, 4H), 1.59 (m, 2H), 1.16 (t,
J=6.01, 3H); ESI-MS m/z 578.37 (M+H).
TABLE-US-00001 TABLE 1 Compound with general formula (I)
composition of the Invention Comp R1 R.sub.2 R.sub.3 4 Et
##STR00026## ##STR00027## 5 Et ##STR00028## ##STR00029## 6 Et
##STR00030## ##STR00031## 7 Et ##STR00032## ##STR00033## 8 Et
##STR00034## ##STR00035## 9 Et ##STR00036## ##STR00037## 10 Et
##STR00038## ##STR00039## 11 Et ##STR00040## ##STR00041## 12 Et
##STR00042## ##STR00043## 13 Et ##STR00044## ##STR00045## 14 Et
##STR00046## ##STR00047## 15 Et ##STR00048## ##STR00049## 16 Et
##STR00050## ##STR00051## 18 ##STR00052## ##STR00053## ##STR00054##
19 ##STR00055## ##STR00056## ##STR00057## 20 ##STR00058##
##STR00059## ##STR00060## 21 ##STR00061## ##STR00062## ##STR00063##
22 ##STR00064## ##STR00065## ##STR00066## 23 ##STR00067##
##STR00068## ##STR00069## 24 ##STR00070## ##STR00071## ##STR00072##
25 ##STR00073## ##STR00074## ##STR00075## 26 ##STR00076##
##STR00077## ##STR00078## 27 ##STR00079## ##STR00080## ##STR00081##
29 ##STR00082## ##STR00083## ##STR00084## 30 ##STR00085##
##STR00086## ##STR00087## 31 ##STR00088## ##STR00089## ##STR00090##
32 ##STR00091## ##STR00092## ##STR00093## 33 Et ##STR00094##
##STR00095## 34 Et ##STR00096## ##STR00097## 35 Et ##STR00098##
##STR00099## 36 Et ##STR00100## ##STR00101## 37 ##STR00102##
##STR00103## ##STR00104## 38 ##STR00105## ##STR00106## ##STR00107##
39 ##STR00108## ##STR00109## ##STR00110## 40 ##STR00111##
##STR00112## ##STR00113##
Experimental Procedure for Screening Toll-Like Receptor 9
Antagonistic Activity
A medium throughput biological assay based on toll-like receptor 9
activation has been designed in primary human immune cells to
screen the synthesized small molecules with general formula (I) for
toll-like receptor 9 (TLR9) antagonism. Among the immune cell
subsets circulating in the peripheral blood, two cell subsets
plasmacytoid dendritic cells (PDCs) and B lymphocytes has
significant expression of TLR9. Plasmacytoid dendritic cells are
capable of producing type I interferons (e.g. IFN-alpha) in
response to TLR9 ligands. Type A and type B unmethylated
cytosine-guanine rich DNA oligonucleotides (CpG oligonucleotides)
are the bonafide ligands for TLR9.
Example 10
The assay is based on the principle that establishes production of
IFN-alpha from human PBMC in response to type A CpG
oligonucleotides (CpGA) almost exclusively results from TLR9
triggering on the PDCs (data not shown). In the screening assay we
isolated PBMCs from venous blood collected from healthy donors
using density gradient centrifugation. PBMCs were cultured the at
2-3*10{circumflex over ( )}5 cells/200 ul/well in a 96 well plate.
TLR9 agonist CpGA was added at 1 uM in presence of escalating doses
of the synthesized small molecules (0 uM, 0.1 uM, 1 uM, 5 uM, 10 uM
and 20 uM). The supernatants was collected after overnight culture
from the culture wells and looked for IFN-alpha using enzyme linked
immunosorbent assay (ELISA). In this screening assay, molecules
having TLR9 antagonistic activity inhibited IFN-alpha production.
The compounds used for the assay and the results were depicted in
FIG. 1. For the biological validation of TLR9 antagonism the bona
fide TLR9 agonist CpG oligonucleotides were used. As no standard
marked small molecule antagonist for TLR9 exists, we did not use
any standard compound.
Example 11
Experimental Procedure for TLR9 Reporter Assay
HEK-Blue-hTLR9 Secreted Alkaline Phosphatase (SEAP) reporter assay
was used to screen compounds with general formula (I) for TLR9
antagonism. Reporter HEK cell lines expressing human TLR9 along
with a NF-.kappa.B promoter driven secreted embryonic alkaline
phosphatase (SEAP) reporter gene were used. A 96 well plate in
complete DMEM medium supplemented with 100 .mu.g/ml Normocin was
used for overnight incubation of 70,000 cells/well at 37.degree. C.
and 5% CO.sub.2. TLR9 agonist CpGB (ODN2006) was added after
incubation to the wells at a concentration of 1 .mu.M in presence
of escalating doses of compounds with general formula (I) and
subsequently incubated for 24 hours at 37.degree. C. and 5%
CO.sub.2. Supernatants were collected and 201 of supernatant was
added to wells containing 200 .mu.l of Quanti-Blue detection media
and further incubated for 2 hours. The OD values were taken at 620
nm in a spectrophotometer. TLR9 antagonistic activity was
calculated based on inhibition of TLR9-mediated NF-kB activation in
a dose-dependent manner (FIG. 2). The compounds used for the assay
and the results were depicted in FIG. 2. For the biological
validation of TLR9 antagonism the bona fide TLR9 agonist CpG
oligonucleotides were used. As no standard marketed small molecule
antagonist for TLR9 exists, we did not use any standard compound.
For specificity the negative control was TLR7-expressing HEK293
reporter cells which were compared to the reporter activity of
TLR9-expressing HEK293 reporter cells.
Example 12
Experimental Procedure for TLR9 Antagonism in Primary Human
pDC.
A medium throughput biological assay was designed based on
toll-like receptor 9 activation in plasmacytoid dendritic cells
(pDC) isolated from PBMCs of healthy donors to screen compounds
with general formula (I). pDCs were isolated from PBMCs by magnetic
immune selection using anti-BDCA4 microbeads. The isolated pDCs
were then cultured at 3*10{circumflex over ( )}4 cells/100
.mu.l/well in a 96 well plate. TLR9 agonist CpGA was added at 500
nM in presence of escalating doses of the compounds with general
formula (I). Supernatants were collected after overnight culture
from the culture wells and looked for IFN-alpha using enzyme linked
immunosorbent assay (ELISA). TLR9 antagonistic activity was
calculated based on inhibition of IFN-alpha production in this
screening assay. The compounds used for the assay and the results
were depicted in FIG. 3. For the biological validation of TLR9
antagonism the bona fide TLR9 agonist CpG oligonucleotides were
used. As no standard marked small molecule antagonist for TLR9
exists, we did not use any standard compound.
Example 13
Experimental Procedure for Screening for Cytotoxicity of the
Identified TLR9 Antagonists
MTT assay is a widely used method for screening drugs and testing
their cytotoxicity. MTT assay is a colorimetric assay for assessing
cell viability. Under defined conditions, NAD(P)H-dependent
cellular oxidoreductase enzymes may reflect the number of viable
cells present. In viable cells with active metabolism,
oxidoreductase enzymes are capable of reducing the tetrazolium dye
MTT 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
to its insoluble purple colour formazan product with an absorbance
maximum near 570 nm. Colour formation serves as a useful and
convenient marker of only the viable cells since when cells die;
they lose the ability to convert MTT into purple colour formazan.
The exact cellular mechanism of MTT reduction into formazan is not
well understood, but likely involves reaction with NADH or similar
reducing molecules that transfer electrons to MTT (FIG. 4). HepG2
(a hepatic epithelial cell line) and SW480 (an intestinal mucosal
epithelial cell line) cells were used to check cytotoxicity of the
compounds with general formula (I). HepG2 and SW480 were cultured
in DMEM Complete media in 96 well plates at density of 30,000 cells
per well, making a final volume of 100 .mu.l/well. Different
concentrations (0.1, 0.5, 1, 10, 20 and 100 .mu.M) of compounds
with general formula (I) was added and subsequently incubated for
24 hours at 37.degree. C. and 5% CO.sub.2 in incubator. To each
well 50 .mu.l of MTT (5 mg/ml) was added and further incubated for
1 to 4 hours at 37.degree. C. Thereafter, 100l of DMSO was added to
each well with proper mixing to ensure complete solubilisation of
formazan crystals. The absorbance was measured at 570 nm using an
ELISA plate reader. TLR9 antagonists with general formula (I) did
not showed any considerable cytotoxicity at concentrations below
100 .mu.M on this assay (FIG. 4). The compounds used for the assay
and the results were depicted in FIG. 4.
TABLE-US-00002 TABLE 2 Depicts IC.sub.50 values of the compounds
with general formula (I) composition of the Invention. Compound
IC.sub.50 Number (.mu.M) 4 0.188 5 0.203 6 0.470 7 0.438 8 0.140 9
1.834 10 0.339 12 0.378 15 0.470 23 0.279 24 0.145 25 0.254 26
0.120 27 0.056 29 0.122 30 0.241 31 0.005 32 0.100 34 0.080 38
8.798
Advantages of the Invention
The synthesized new compounds with general formula (I) of the
present invention have several advantages. 1. The compounds with
general formula (I) can effect immune stimulation via TLR9
antagonism. 2. The compounds with general formula (I) are capable
of inhibiting immune stimulation mediated through TLR9. 3. The
compounds with general formula (I) can be evaluated by medium
throughput biological assays involving human peripheral blood
mononuclear cells, isolated human primary pDCs and reporter assay
using transfected TLR9 cells. The assay system was standardized and
the results from all three assay systems can be correlated. 4. The
compounds with general formula (I) can be used in a number of
clinical contexts for treating conditions involving unwanted immune
activity in response to a suitable TLR ligand or TLR signalling
agonist where inhibition of TLR9 mediated signalling is
important.
* * * * *